Helicobacter pylori

1.5. Helicobacter pylori

1.5.1. History, prevalence, and transmission

The human gastric pathogen Helicobacter pylori colonizes the stomach and shares a 100 000-year old history with its human host. Together with the modern human being, H. pylori spread from East Africa around the world approximately 58 000 years ago (Linz et al. 2007). In 1984 Berry Marshall and Robin Warren discovered and characterized H. pylori in human gastric biopsies and were awarded in 2005 with the Noble Prize in physiology and medicine for the link of gastritis to be caused by H. pylori (Marshall and Warren 1984; Marshall et al. 1985). The bacterium descended from the genus Campylobacter and was named as such until Goodwin, McCullough, and Boehm (1989) classified and renamed into Helicobacter pylori. H. pylori is the causing agent for gastric disorders like gastritis which evolves asymptomatic in most of the cases and a chronic infection can induce peptic ulcer disease (10-20 %), distal gastric adenocarcinoma (1-2 %), and gastric mucosal-associated lymphoid tissue (MALT) lymphoma (< 1 %) (Bauer and Meyer 2011). Due to its strong correlation with gastric cancer, H. pylori was declared in 1994 by the International Agency for Research on Cancer as class I human carcinogen (Parsonnet et al. 1991; IARC 1994). Although showing a declining incidence rate, gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer-related death after lung cancer, with 75 % of the cases attributable to H. pylori (de Martel, Forman, and Plummer 2013; de Martel et al. 2012). The development of gastric cancer is sequelae of steps that are described by Correa’s cascade (Figure 6), always starting from chronic gastritis due to persistent inflammation as the major cause of gastric cancer. Chronic gastritis develops further into multifocal atrophic gastritis, intestinal metaplasia, dysplasia and finally into invasive adenocarcinoma and gastric cancer (Correa and Piazuelo 2012; Correa et al.

1975).

1.5 Helicobacter pylori

Figure 6: Schematic representation of H. pylori-induced Correa's cascade. The infection of the normal gastric mucosa with H. pylori induces chronic gastritis which consecutively develops further into atrophy, intestinal metaplasia, dysplasia, and intestinal-type gastric cancer. Bacteria are not detected anymore in the stage of intestinal metaplasia. The figure was adapted and modified from Correa and Piazuelo (2012).

Overall about 50 % of the world’s population is infected with H. pylori while the prevalence is higher in developing countries (80 %) e.g. Saudi Arabia, India and Vietnam than in industrialized countries (< 40 %) due to lower socioeconomic statuses like housing conditions and hygiene. Infections are mostly acquired during childhood and can persist, if not treated through the whole lifetime of the host (Peek and Blaser 2002; Hooi et al. 2017). Nevertheless, only a minority of H. pylori infections lead to gastric cancer, but an infection increases the risk significantly (Peek and Blaser 2002).

The transmission of H. pylori is still not fully understood. Most probably H. pylori are transmitted via the gastro-oral or fecal-oral route from human to human, but also transmitted through contaminated food and water was reported (Bauer and Meyer 2011). The treatment for H. pylori eradication is a standard triple therapy consisting of a proton pump inhibitor, clarithromycin and amoxicillin or metronidazole, but the efficacy of this treatment is decreasing because of H. pylori resistance against the antibiotics clarithromycin as well as metronidazole (Papastergiou, Georgopoulos, and Karatapanis 2014). Due to the co-evolution of the pathogen and the human host, H. pylori is highly adapted to its host to develop a persistent infection.

1.5.2. Biology of H. pylori

Helicobacter pylori is a Gram-negative, spiral-shaped bacteria with two to six unipolar flagella which allow the high motility of this bacterium, required for the penetration of the thick mucus layer (Goodwin, McCullough, and Boehm 1989; Suerbaum, Josenhans,

1.5 Helicobacter pylori and Labigne 1993). Besides the spiral shape, also a coccoid form occurs which was described to be a non-replicative form of H. pylori and an early stage of cell death (Kusters et al. 1997). H. pylori requires microaerophilic growth conditions at neutral pH and to overcome the acidic pH of the stomach H. pylori expresses urease to neutralize the low pH in the microenvironment (Kusters, van Vliet, and Kuipers 2006).

Interestingly, only 20 % of H. pylori in the gastric mucosa adhere to epithelial cells.

Different autotransporter proteins, so-called adhesins like BabA, SabA, OipA or HopZ are expressed by H. pylori and mediate the adhesion to the epithelial cell surface (Bauer and Meyer 2011). H. pylori are equipped with a panel of virulence factors. Amongst others, these are the vacuolating toxin A (VacA) and the cag pathogenicity island (cagPAI) encoding the effector protein cytotoxin-associated gene A (CagA). Both VacA and CagA are strongly associated with the development of gastric cancer and peptic ulcer disease (Cover and Blaser 2009). Only 60-70 % of the western H. pylori strains harbor cagPAI compared to almost 100 % occurrence in Asian strains (Bauer and Meyer 2011). The cag pathogenicity island not only encodes CagA but also a Type IV secretion system (T4SS) to inject CagA into the cytoplasm of the host cell. The oncogenic feature of CagA was shown in vivo in mice and zebrafish where the expression of the protein alone induced the development of gastric adenocarcinoma (Ohnishi et al. 2008; Neal et al. 2013). In the cytosol, CagA is phosphorylated by host cell tyrosine kinases (c-Src and c-Abl) at the EPIYA (Glu-Pro-Ile-Tyr-Ala) motif (Mueller et al. 2012). The phosphorylation leads further to the activation of multiple host signaling pathways regulating inflammation, cell proliferation or inhibition of apoptosis (Backert and Blaser 2016). In vitro phosphorylated CagA activates the host phosphatase SHP2 inducing a cell elongation also described as the hummingbird phenotype (Higashi et al. 2002). The pore-forming toxin VacA is expressed in all H. pylori strains with allelic variations, determining the cytotoxic severity. The induced vacuolation in the host cell has effects on endocytic compartments and mitochondria by triggering the apoptotic cascade. Besides epithelial cells, VacA has also an influence on immune cells which can be immunostimulatory or immunosuppressive (Kim and Blanke 2012). Furthermore, H. pylori are able to escape the host immune response by depleting cholesterol from the host cell membrane and thereby prevent the activation of the pro-inflammatory interferon 𝛾 (INFG) signaling pathway (Morey et al. 2018).

1.6 Aim of the study

1.6. Aim of the study

The human gastric mucosa is a complex structure organized in gastric units, referred to as glands formed by the epithelium, the lamina propria with gastric stromal cells and the muscularis mucosae a layer of myofibroblasts. The gastric units are populated by spatially distributed specialized cells with distinct functions, including parietal cells, chief cells, and foveolar cells, all deriving from a common stem cell progenitor. The cell-type composition of the gastric glands changes along the proximal-distal axis of the stomach, with oxyntic glands in the corpus and mucus glands in the antrum. From other tissues such as the intestine, it is known that the stromal cells surrounding the crypt communicate and interact with the adjacent epithelium, and recent studies revealed similar interactions between stroma and epithelium in the gastric mucosa, although the knowledge is still limited. Thanks to lineage tracing experiments in mice there is an increasing understanding of the stem cell niche in the gastric mucosa, however very little is known about the differentiation dynamics and the niche factors guiding the differentiation of stem cells into the specialized cells types. Most of the obtained data about the physiology and the patterning of the gut were generated in mice; therefore the transferability of the observations to the human physiology is still uncertain. 3D culture models as represented by organoids constitute a promising alternative to murine experimentation for understanding the complex biology of epithelial cells. A major drawback, however, is the short growth period of a single passage as organoids which is a disadvantage regarding the performance of long-term studies and the assessment of tissue homeostasis. In this thesis, it was aimed to establish an alternative long-lived in vitro culture model of human gastric primary cells that enables long-term cultivation based on the preservation of the stem cells to study tissue homeostasis as well as the study of the responses to an infection with the pathogen Helicobacter pylori.

Accordingly, the presented work is structured in three parts:

The objective of part I is to develop a stable and reliable in vitro culture model for human primary gastric epithelial cells that represent the in vivo situation and would thus allow analyzing the mucosal homeostasis under physiologic conditions. This part was done in collaboration with Dr. Francesco Boccellato. The herein developed “mucosoid

1.6 Aim of the study culture” model was successfully applied in part II to understand the luminal-basal axis patterning of the corpus gland. Accordingly, the differentiation dynamics and signaling niche factors of the oxyntic gland inducing the differentiation of the three major specialized cell types’ foveolar cells, parietal cells, and chief cells were explored. The results obtained in vitro could further be validated ex vivo and in situ using human tissue specimens.

The focus of part III was on analyzing the communication between human gastric stromal cells of the lamina propria and gastric epithelial cells. Therefore human primary stromal cells were to be isolated from the same tissue as the gastric epithelial cells in order to establish a co-culture model of both cell types using the mucosoid culture model. The intention of this work was to obtain an understanding of the paracrine communication of stromal cells with epithelial cells under physiologic conditions but also under pathophysiologic conditions during the course of an infection with Helicobacter pylori and to assess whether stromal cells contribute to the inflammatory response against epithelial infection.

2.1 Material