Transcriptome analysis of the challenged gut barrier in rats Mucosal response to Salmonella and Fructo-oligosaccharides Wendy Rodenburg Promotor: Prof. dr. M.B. Katan Hoogleraar Voedingsleer Instituut voor Gezondheidswetenschappen Vrije Universiteit, Amsterdam Tot 2006: Persoonlijk hoogleraar Afdeling Humane Voeding, Wageningen Universiteit Co-promotoren: Dr. J. Keijer Senior wetenschappelijk onderzoeker Cluster Bioactieve Stoffen RIKILT - Institute of Food Safety, TI Food and Nutrition Dr. ir. I.M.J. Bovee-Oudenhoven Projectleider TI Food and Nutrition, NIZO Food Research Promotiecommissie: Prof. dr. M. Müller Wageningen Universiteit Prof. dr. J. Wells Wageningen Universiteit Prof. dr. R.J. Brummer Universiteit Maastricht Prof. dr. T.A. Niewold Katholieke Universiteit Leuven, België Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG (Voeding, Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid) Transcriptome analysis of the challenged gut barrier in rats Mucosal response to Salmonella and Fructo-oligosaccharides Wendy Rodenburg Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op dinsdag 5 februari 2008 des namiddags te vier uur in de Aula. Transcriptome analysis of the challenged gut barrier in rats; mucosal response to Salmonella and Fructo-oligosaccharides Wendy Rodenburg Thesis Wageningen University (2008), the Netherlands - with summary in Dutch ISBN 978-90-8504-870-1 Abstract Introduction The gut barrier protects the body against harmful substances and microbes. It consists of the gut mucosa, the immune system and the microflora. Crosstalk between these elements determines the mucosal response to stresses. In this thesis, we have studied how the gut mucosa in a living organism reacts to two stress stimuli: Salmonella and fructo-oligosaccharides (FOS). For this we used transcriptome analysis. Results Changes in detoxification, glucose, lipid, peptide and ion transport and proteolysis were part of the early gene expression responses of the rat intestinal mucosa. Neither Salmonella nor FOS altered the expression of barrier related genes such as tight junction, mucin or toll like receptor genes. In contrast, energy metabolism was clearly affected by FOS and could be responsible for the increased permeability induced by FOS. The gene expression response to Salmonella in rats was subtle, this differs from the responses observed in cell culture studies. In contrast to the general expectation, the colon was as much a target for Salmonella as the ileum. FOS increased the expression of Salmonella induced genes, including defence genes, coinciding with increased Salmonella infection. This showed that increase in defence genes reflects a reduced rather than improved gut barrier function, as is often assumed. Several new candidate biomarker genes were identified, such as pancreatitis associated protein (Pap), lipocalin, calprotectin and phospholipase A2. PAP protein was studied in more detail. Its response dependent excretion suggests that it can potentially be used as a non invasive marker. Finally, based on the difficulties encountered in analysing transcriptomic data, we propose a framework to identify biologically relevant genes. Conclusion We identified biological processes not earlier associated to gut barrier functioning. Expected barrier processes were not induced, thus gut barrier research should not focus on expected barrier processes alone. Extrapolating data from model systems of the barrier to the intact animal should be done with great care, as overlap in gene expression is low. Transcriptome analyses have significantly increased the understanding of the actual in vivo barrier processes and have delivered potential new gut health biomarkers. - 5 - - 6 - Content Abstract 5 Chapter 1 General introduction 9 Chapter 2 Gene expression response of the rat small intestine following oral Salmonella infection 27 Chapter 3 Salmonella induces prominent gene expression in the rat colon 49 Chapter 4 Ileal mucosal and faecal pancreatitis associated protein is increased during Salmonella infection in rats and is associated with infection severity 75 Chapter 5 A framework to identify physiological responses in microarray based gene expression studies: selection and interpretation of biologically relevant genes 89 Chapter 6 Impaired intestinal barrier function by dietary fructo- oligosaccharides (FOS) in rats is associated with increased mitochondrial gene expression 113 Chapter 7 General discussion 135 Samenvatting 155 Supplemental data 161 Dankwoord 175 About the author 179 - 7 - Chapter 1 General introduction 1. Introduction 2. Intestinal mucosal barrier 2.1 The epithelium 2.2 The immune system 2.3 The microflora 3. External factors affecting the gut barrier 3.1 Intestinal bacterial infections 3.2 Nutritional modulation of intestinal epithelium 4. Transcriptome analysis 4.1 Transcriptomic technique 4.2 From genes to mechanisms 4.3 Intestinal genomics 5. Outline of this thesis - 9 - Chapter 1 1 Introduction The primary function of the intestinal epithelium is to digest and absorb nutrients. At the same time it has to prevent infiltration of pathogens and harmful compounds. This barrier function is important for an organism’s health and is tightly controlled. Disturbance can be caused by pathogens, harmful compounds or nutrients, and may lead to diarrhoea, infectious disease or uncontrolled inflammation38. Reduction of early or mild disturbances could prevent the onset of harmful inflammation reactions. However little is known about the early processes involved in disturbances of the intestinal epithelium. Understanding of the epithelial barrier responses is limited to model systems such as in vitro cell cultures, or ex vivo epithelial tissue cultures. These models cannot provide a full overview of the multifactorial in vivo situation, where the luminal content, the gut microflora and the gut immune system influence the responses of the intestinal epithelium. Therefore, to obtain an overview of the early responses of the intestinal epithelium, examination of the in vivo situation is required. Examination of gut barrier mechanisms is possible only after challenging the barrier through induction of stress. This indicates whether the gut barrier is able to resist the stress or not, and it reveals which processes appear to be necessary to resist the stress. In this study, we chose two types of challenges: a pathogenic bacterial challenge and a dietary challenge. Dietary components can affect the intestinal epithelium directly or indirectly, via changes in the intestinal contents or changes in the endogenous microflora. Human15 and animal16,103 studies have shown that diet can modulate intestinal infections. For example, Salmonella infection is sensitive to dietary modulation by calcium and by fructo-oligosaccharides (FOS). Calcium decreases colonization and translocation of Salmonella, whereas FOS increases translocation of this pathogen in rats. However the Salmonella-induced biological processes in the intestinal mucosa and the possible dietary modulation of these processes are not known. DNA microarray technology allows thousands of genes to be studied at the same time and has been successful in identifying in vivo molecular responses of intestinal tissues to commensal bacteria, pathogens or nutrients111,112. This technique is not restricted to a priori defined biological processes, but identifies all processes active at the time of examination. The aim of this thesis research was to identify the early gene expression response of the intestinal mucosa in rats to two challenges that adversely affect the barrier function: Salmonella and FOS. We used transcriptomic analysis to look at the whole genome. Increased insight into the molecular response of the gut barrier allows monitoring of gut health and the development of nutrients or pharmaceuticals that are able to modulate early mucosal responses and improve intestinal resistance, for example to infectious disease. 2 Intestinal mucosal barrier To provide optimal nutrient absorption, the mucosal surface of the intestinal tract is large. In adult humans it represents a surface area of approximately 200m2, which is 100 times larger than the surface of the skin. In rats, the gut surface is 25 times larger than the skin surface25. This large - 10 - Introduction surface area, which is in constant contact with the external environment, makes the intestinal mucosa an important target for harmful compounds, such as pathogenic microorganisms, toxins or harmful nutrients. Fortunately, the gastro intestinal (GI) track is equipped with several mechanisms that prevent survival of ingested microorganisms. For example, the acidic environment of the stomach39, bile117 and pancreatic enzymes of the small intestine88, and motility in the small intestine93. These mechanisms prevent survival and colonization of microorganisms in the intestinal lumen. Despite these mechanisms, the intestinal epithelium is constantly exposed to unwanted compounds. Successful resistance to these compounds is possible due to the intestinal mucosal barrier. This barrier consists of a monolayer of epithelial cells, the mucosal immune system and the microflora (figure 1). These three components are in continuous interaction with each other. Pathogens Microflora Mucus layer Epithelial monolayer Immune cells Figure 1. The intestinal barrier aligns the entire gastro-intestinal system. 2.1 The