Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Microbiology & Immunology
Non Technical Summary
This study is focused on He/icobacter suis, a bacterial zoonotic pathogen that causes gastric inflammation in both humans and domestic pigs. Our preliminary data obtained by screening slaughterhouse samples from Big Timber, MT, revealed that a significant proportion of Montana pigs are infected with H. suis, and that infection is associated with gastric pathology, consistent with earlier studies. We here propose to analyze additional pig stomach tissue samples for the presence of infection and pathology in order to obtain reliable data on the prevalence of this infection. We will also perform ex vivo and in vitro analyses to characterize the inflammatory and immune response that H. suis causes in the pig and human stomach. These data will fill an important knowledge gap in our understanding of H. suis pathogenesis and will allow us to better estimate the risk of human H. suis transmission and disease.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
H. suis infection is associated with gastric pathology in humans, pigs, and other domestic and laboratory animals. However, little is known about how H. suis bacteria interact with the host to cause inflammation and pathology. We hypothesize that H. suis causes gastric disease in pigs and humans by inducing pro-inflammatory responses in gastric epithelial cells. We will test our hypothesis by addressing thefollowing objectives:Objective 1: Determine the level of infection with H. suis in Montana pigs and elucidate the association of H. suis infection with gastric disease. To analyze whether H. suis causes gastric disease in Montana pigs, we will screen slaughterhouse samples of gastric body and antrum for the presence of H. suis infection and gastric pathology. Specifically, H. suis infection will be determined by quantitative PCR and rapid urease CLO test, and tissue sections from gastric antrum and the gastric body will be analyzed microscopically for inflammatory alterations and the presence of spiral-shaped bacteria. The presence of H. suis in select samples will be confirmed by microbial sequencing. We will also collect data on pig breeds, husbandry methods, and carcass weights for possible correlation with infection rates and pathology. These experiments will reveal the disease burden of H. suis gastritis in Montana pigs.Objective 2: Determine the mechanisms by which H. suis drivesepithelial inflammatory responses using a human gastric organoid model. The gastric epithelium is the first point of contact between Helicobacter bacteria and the host. In the infected human stomach, immune cells including neutrophils, macrophages and dendritic cells are attracted to the sites of infection by chemokines and cytokines released from the epithelium, in particular IL-8. In preliminary experiments, we showed that H. suis induced gastric epithelial IL-8 expression in human organoids, although H. suis lacks the Gag pathogenicity island that is the major driver of IL-8 secretion induced by H. pylori (Vermoote et al. 2011). Here, we will analyze the molecular mechanisms by which H. suis induces epithelial cell inflammation. We will use human gastric organoids to analyze (1) epithelial proinflammatory cytokine and chemokine production in response to H. suis, (2) NF-KB activation in response to H. suis, and (3) signaling pathways involved in NF-KB activation. Responses to H. suis will be compared to responses to H. pylori, the most common human gastric pathogen.Objective 3: Establish a porcine gastric organoid model to analyze the inflammatory epithelial cell response of porcine gastric epithelial cells to H. suis. A recent report has highlighted significant differences in the immune response to H. suis infection in different species (Bosschem et al. 2017).Therefore, we propose to compare the response of H. suis-infected porcine gastric epithelial cells to that of human gastric epithelial cells. Based on our experience with human gastric organoids, we will establish gastric organoid lines from the porcine stomach. As described for Objective 2 above, porcine gastric organoids will be infected with H. suis bacteria in vitro, and cytokine expression, NF-KB activation and induction of cell death will be determined.
Project Methods
Objective 1: Determine the level of infection with H. suis in Montana pigs and elucidate the association of H. suis infection with gastric disease.1.1. Analyze porcine gastric tissue samples for the presence of H. suis infection and gastric pathology. Porcine tissue samples for analysis of H. suis infection will be obtained from a commercial slaughter operation in Big Timber, MT. Data on carcass weight, farm of origin and husbandry system will be provided. We will analyze tissue collected from the pyloric antrum and the glandular part of the gastric body for H. suis infection by rapid urease CLO Test, and for H. suis urease-specific sequences by quantitative PCR. To confirm the specificity of the PCR detection, PCR products for H. suis urease genes from a subset of samples will be sequenced. In addition, paraffin-embedded sections obtained from gastric antrum and body will be analyzed for the presence of spiral bacteria and scored for histopathological changes. We have already collected samples from 100 pigs and will continue to analyze these samples and collect additional tissues until matched CLO-test, qPCR and histopathological analyses have been performed on 100 - 150 samples. Pearson's correlation coefficient will be calculated to determine a relationship between H. suis copy numbers and pathology scores. 1.2. Using immunofluorescence microscopy, characterize the cellular infiltrates induced by H.suis infection of the porcine stomach. H. pylori infection of the human stomach leads to chronic active gastritis with initial polymorphonuclear infiltrates and increased numbers of lymphocytic infiltrates and lymphoid follicles seen at later stages. In contrast, infiltrates associated with porcine H. suis infection have been characterized as mostly lymphocytic, with a large number of plasma cells present. However, these results were exclusively based on tissue staining with H&E. Here, we will analyze gastric tissue samples from H. suis-infected and non-infected pigs for mucosal infiltration with mononuclear phagocytes, T cells, B cells and neutrophilic granulocytes by immunocytochemistry, using antibodies to SLA-DQ, CD3, CD79a and SWC8, respectively. Antibody binding will be visualized using appropriate horse radish peroxidase-labeled secondary antibodies and DAB as a substrate. Slides will be analyzed on an Olympus BX60 upright fluorescence microscope, and cellular infiltrates will be quantified by digital image analysis.Objective 2: Determine the mechanisms by which H. suis drives epithelial inflammatory responses using a human gastric organoid model2.1. Determine whether H. suis induces NF-κB signaling in primary human gastric epithelial cells through the engagement of NOD1, TLR2 and/or CD74. To analyze the inflammatory response of primary human gastric epithelial cells to H. suis, we will use gastric organoid cultures derived from human tissue samples generated and maintained as described in our recent publication (Sebrell et al., 2018). We will infect the gastric organoids with H. suis DSM 19735T, or with H. pylori as a positive control. To infect epithelial cells, organoids will be re-seeded on collagen-coated plates to obtain confluent monolayers and bacterial will be added to the plates. We will then analyze NF-κB activation by microscopy to determine nuclear translocation and by phosflow FACS analysis to determine p65 phosphorylation. CagA-positive and CagA PAI-deficient H. pylori mutants will be used to determine the contribution of CagA signaling to H. pylori-induced NF-κB-activation. If we can show that H. suis activates NF-κB, we will next determine whether NOD1, TLR2 or CD74 are involved in the activation pathway. TLR2 and CD74 will be blocked using neutralizing antibodies, and NOD1 will be blocked using the small molecule inhibitor ML130. Cells will then be treated with H. suis or H. pylori and analyzed for NF-κB activation as described above. Having shown that H. suis drives IL-8 expression, we anticipate that H. suis infection also results in NF-κB phosphorylation and nuclear translocation.2.2. Determine whether H. suis induces gastric epithelial cell release of pro-inflammatory cytokines to initiate gastric inflammation. As shown in preliminary experiments, H. suis caused increased IL-8 gene expression in primary human gastric epithelia. To confirm that H. suis infection causes secretion of IL-8 and other pro-inflammatory cytokines, human gastric organoids will be re-seeded as monolayers on collagen-coated transwell inserts. The gastric epithelial cells will then be treated with live H. suis, H. suis lysates, live H. pylori, or will be left untreated. We will culture supernatants for cytokine secretion using a cytometric bead array. In parallel, cultured cells will be analyzed for H. suis-induced cell death using Annexin/PI. Morphological alterations of the epithelial cells will be monitored by microscopy. We hypothesize that H. suis and H. pylori infection will induce gene expression and secretion of IL-8 and other epithelial cell associated pro-inflammatory cytokines, particularly IL-1β and IL-6. However, since virulence factor expression differs between H. suis and H. pylori, we do anticipate to find differences in the magnitude of the response and the cytokine profile.Objective 3: Establish a porcine gastric organoid model to analyze the inflammatory epithelial cell response of porcine gastric epithelial cells to H. suis.3.1. Optimize the establishment and culture of porcine gastric organoids from antrum and gastric body. In our preliminary experiments, we were able to establish organoid cultures from porcine stomach using our human gastric organoid protocol. However, efficiency of spheroid formation and growth rates of the organoids were very low. Here, we seek to improve our protocol for porcine gastric organoid generation and maintenance. We will compare our published collagenase digestion protocol to gland isolation protocols that involve EDTA treatment alone and to a protocol that utilizes a commercially available dissociation reagent. Second, we will optimize our growth medium by varying medium concentrations of growth factors Wnt3a, R-spondin, ROCK inhibitor and the TGF-β inhibitor and supplementation of media with additional supplements such as epidermal growth factor and niacin that have been used by others to maintain optimal growth of gastric organoids. Organoid formation and growth under different conditions will be monitored by live imaging microscopy, and epithelial cell differentiation will be confirmed by RT-PCR analysis. We anticipate that by adjusting our gland isolation and organoid culture protocols, we will successfully establish multiple porcine gastric organoid lines for in vitro modeling of H. suis infection.3.2. Determine whether H. suis infection of porcine gastric organoids induces pro-inflammatory epithelial cell responses and epithelial cell death. To determine the epithelial response of the porcine gastric epithelium to H. suis, we will analyze porcine gastric organoids for NF-κB activation, pro-inflammatory cytokine expression and cell death. As described for Objective 2, Exp. 1, we will infect the porcine organoids with H. suis and analyze them for NF-κB activation. If we can successfully show that H. suis activates the pro-inflammatory transcription factor NF-κB in the porcine gastric epithelium, we will next analyze H. suis-induced proinflammatory cytokine expression using quantitative RT-PCR. If we can determine increased expression of any of the cytokines by qRT-PCR, we will confirm these data by performing ELISA analyses of culture supernatants. We will also analyze the infected porcine gastric organoid cells for epithelial cell death. These experiments will provide initial insights into the pro-inflammatory response of the porcine gastric epithelium to de novo H. suis infection.