The human microbiome in Barrett’s esophagus is hard to stomach

The incidence of esophageal adenocarcinoma (EAC) has increased nearly five-fold over the last four decades in the United States. Barrett's esophagus, the replacement of the normal squamous epithelial lining with a mucus-secreting columnar epithelium, is the only known precursor to EAC. Like other parts of the gastrointestinal (GI) tract, the esophagus hosts a variety of bacteria and comparisons among published studies suggest bacterial communities in the stomach and esophagus differ. Chronic infection with Helicobacter pylori in the stomach has been inversely associated with development of EAC, but the mechanisms underlying this association remain unclear.The bacterial composition in the upper GI tract was characterized in a subset of participants (n=12) of the Seattle Barrett's Esophagus Research cohort using broad-range 16S PCR and pyrosequencing of biopsy and brush samples collected from squamous esophagus, Barrett's esophagus, stomach corpus and stomach antrum. Three of the individuals were sampled at two separate time points. Prevalence of H. pylori infection and subsequent development of aneuploidy (n=339) and EAC (n=433) was examined in a larger subset of this cohort.Within individuals, 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. Re-sampling of study participants revealed similar upper GI community membership in two of three cases. In this Barrett's esophagus cohort, Streptococcus and Prevotella species dominate the upper GI and the ratio of these two species is associated with waist-to-hip ratio and hiatal hernia length, two known EAC risk factors in Barrett's esophagus. H. pylori-positive individuals had a significantly decreased incidence of aneuploidy and a non-significant trend toward lower incidence of EAC

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

Thesis (Ph.D.)--University of Washington, 2018Helicobacter 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

Autoimmunity Initiates in Nonhematopoietic Cells and Progresses via Lymphocytes in an Interferon-Dependent Autoimmune Disease

The type I interferon (IFN) response initiated by detection of nucleic acids is important for antiviral defense but is also associated with specific autoimmune diseases. Mutations in the human 3′ repair exonuclease 1 (Trex1) gene cause Aicardi-Goutières syndrome (AGS), an IFN-associated autoimmune disease. However, the source of the type I IFN response and the precise mechanisms of disease in AGS remain unknown. Here, we demonstrate that Trex1 is an essential negative regulator of the STING-dependent antiviral response. We used an in vivo reporter of IFN activity in Trex1-deficient mice to localize the initiation of disease to nonhematopoietic cells. These IFNs drove T cell-mediated inflammation and an autoantibody response that targeted abundant, tissue-restricted autoantigens. However, B cells contributed to mortality independently of T cell-mediated tissue damage. These findings reveal a stepwise progression of autoimmune disease in Trex1-deficient mice, with implications for the treatment of AGS and related disorders. [Display omitted] ► Trex1 is an essential negative regulator of the STING-dependent antiviral response ► Nonhematopoietic cells initiate IFN-dependent autoimmunity in Trex1-deficient mice ► T cells are necessary and sufficient for autoimmune inflammation in Trex1−/− mice ► B cells contribute to mortality in Trex1−/− mice independently of inflammatio

Pathway-Focused Arrays Reveal Increased Matrix Metalloproteinase-7 (Matrilysin) Transcription in Trachomatous Trichiasis

A method of identifying genes with significantly altered expression in persons with trichiasis is described

The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage

<i>Streptococcus</i> to <i>Prevotella</i> species ratio corresponds to phylogenetic distance sample clustering and correlates with Barrett’s esophagus risk factors.

<p>(A) Cluster analysis of KR distances between microbial communities of individual study samples. Pyroseq. <i>Strep</i>:<i>Prev</i> ratio was calculated using relative abundance of mapped reads for all <i>Streptococcus</i> and <i>Prevotella</i> species as determined by pyrosequencing. ddPCR <i>Strep</i>:<i>Prev</i> ratio was calculated using copies/μl of a <i>Streptococcus</i> or <i>Prevotella</i>-specific 16s rRNA gene segment as determined by droplet digital PCR. Samples color-coded based on the majority of calculated Pyroseq. <i>Strep</i>:<i>Prev</i> ratios in a group being <0.5 (blue), 0.5–1.5 (green), 1.5–4.0 (magenta) or >4.0 (red). (B) Boxplots comparing <i>Streptococcus</i> to <i>Prevotella</i> ratio as determined by pyrosequencing and ddPCR. The central line within each box represents the median of the data, the whiskers represent the 5^th and 95^th percentiles and data outside that range are plotted as individual points. (C) Relationship of <i>Streptococcus</i> to <i>Prevotella</i> ratio (measured by ddPCR) and waist-hip ratio of all male participants segregated by anatomic site. Strength of association between these two variables was determined by Pearson’s correlation test with correlation coefficient squared (r²) values as indicated. (D) Relationship of <i>Streptococcus</i> to <i>Prevotella</i> ratios (measured by ddPCR) and hiatal hernia length in all participants segregated by anatomic site. Strength of association tween these two variables was determined by Pearson’s correlation test with correlation coefficient squared (r²) values as indicated.</p

Members of the <i>Firmicutes</i> or <i>Bacteroidetes</i> phyla dominate the upper gastrointestinal tract microbiome.

<p>(A) Phylogenetic relationship of the top 45 OTUs recovered from each of the four sites sampled in individual participants. Respective phyla are noted above main branches of the phylogenetic tree. Numbers in parentheses represent total number of pyrosequencing reads recovered for a given species or genera across all samples followed by the fraction of participants in whom a relative abundance of ≥1.3% of a given species or genera were detected. (B) Species/genera-level profiles of top 45 OTUs detected by 454 sequencing in squamous esophagus, Barrett’s esophagus, stomach corpus and stomach antrum of indicated participants. Data arranged in order of increasing <i>Firmicutes</i> dominance. Individual species/genera are color-coded according to scheme presented in (A). Sequencing reads from brush samples were used when available, otherwise, data from biopsy samples are shown. Species reads were normalized to the total number of reads per corresponding site in a given individual. ^†Denotes samples collected at a second time point (P2 [t = 4 months]; P7 [t = 2 years]; P9 [t = 3 years]); <i>Hp+</i> indicates <i>H</i>. <i>pylori</i>-positive individual. Italicized participant IDs denote data from biopsy samples in cases where brush samples were not available for analysis.</p

Participant Demographics.

<p>^a LG = low grade HG = high grade</p><p>^b Denotes samples collected at a second time point (P2 [t = 2 years]; P7 [t = 4 months]; P9 [t = 3 years])</p><p>^c Denotes <i>H</i>. <i>pylori</i>-positive participant</p><p>Participant Demographics.</p

Upper gastrointestinal microbiome similarity with replicate sampling.

<p>(A–C) Species/genera-level profiles of microbiota detected by 454 sequencing in squamous esophagus, Barrett’s esophagus, stomach corpus and stomach antrum of individuals P7 at the time of first [t = 0] and second sample collection [t = 4 months] (A), P2 at t = 0 and t = 2 years (B) and P9 at t = 0 and t = 3 years (C). Individual species/genera are presented according to coloring scheme described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129055#pone.0129055.g002" target="_blank">Fig 2</a> (D) Phylogenetic KR distance between (inter) samples from participants P2, P7 and P9 at both time points and within those individuals comparing the 1^st and 2^nd time points from the indicated anatomic site. The central line within each box represents the median and the whiskers represent the minimum and maximum values.</p