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The gastrointestinal system is under constant antigenic challenge from huge numbers of microbes. The gastrointestinal mucosal surface is a portal entry to a large numbers of foreign antigens including those from bacteria. The continuous exposures of the gastrointestinal mucosa to large numbers of bacteria have made the mucosal immune system evolve mechanisms to elicit different responses to pathogenic bacteria and commensal bacteria. Both commensal and pathogenic bacteria are recognized by the host's pattern recognition molecules, including TLR and NLR. Although commensal and pathogenic bacteria have common conserved microbial motifs that can activate the pattern recognition molecules, the different outcome of pathogen-host interaction from non-pathogen-host interaction is the activation of strong innate and adaptive immune responses directed at eradicating the pathogens (as reviewed by (Magalhaes et al., 2007) and (Salzman et al., 2007)
Mucosal epithelial cells lining the gastrointestinal tract provide more than a mere physical barrier to infection. Mucin glycoproteins produced by mucus-producing cells in the epithelium or submucosal glands are an integral part of the mucosal barrier to pathogens (as reviewed by (Linden et al., 2008b)). MUC1 and MUC13 belong to cell surface mucin family, and have an important role in modulating immune responses against bacteria in the stomach and intestine, respectively (Williams et al., 2001, McAuley et al., 2007, McGuckin et al., 2007). Following the invasion of the mucus layer by bacteria, the large extracellular domain of cell surface mucins can act as releasable decoy ligands for bacterial adhesions, thereby preventing subsequent bacterial attachment to and invasion of epithelial cells (McAuley et al., 2007, Every et al., 2008, Linden et al., 2009). Moreover, cell surface mucins initiate intracellular signalling that may be important as sensor mechanisms in response to invasion or epithelial damage (as reviewed by (Carraway et al., 2003)).
It was shown that mice lacking MUC1 are predisposed to more severe pathology following Helicobacter pylori infection (McGuckin et al., 2007) and Campylobacter jejuni infection (McAuley et al., 2007). However, crosstalk between mucins and TLR and NOD signalling in the context of anti-inflammatory activity of mucins in response to pathogens is of potential importance in determining the nature of the inflammatory response to infection.
The Gastrointestinal Immune System against Pathogenic Bacteria
Distinguishing between Commensal and Pathogenic Bacteria
The mucosal surfaces of the gastrointestinal tract are vulnerable to infection due to their anatomical and physiological properties. They are thin and permeable which allow food absorption. This permeability creates vulnerability to infection. However, even though the gastrointestinal milieu is colonized by a dynamic microbial ecosystem, the vast majority of these microbes do not cause infection and disease. The mucosal immune system has evolved mechanisms to elicit different responses to pathogenic bacteria and commensal bacteria. On one hand, the gastrointestinal immune system is required to avoid a vigorous immune response to food antigens or commensal bacteria, whilst it is obliged to detect and kill pathogens reaching the gastrointestinal mucosal surface (as reviewed by (Magalhaes et al., 2007) and (Blum and Schiffrin, 2003)).
Despite the continuous exposure of bacteria in close proximity to the intestinal surface epithelium, it is very unusual that the commensal bacteria cause disease. The host's immune system has several mechanisms to control the immune response towards the commensal bacteria. Being recognized by TLRs and NODs, then the bacteria are sampled by dendritic cells. The sampling by dendritic cells occurs in M cells that overlay the intestinal Peyer's patches and lymphoid follicles or in the epithelial junctional complexes. Special subsets of the dendritic cell population (CD11c+CD11b+CD8ï¡-) are responsible for maintaining tolerance to harmless bacteria by inducing Th2-induced cytokines and IL-10, leading to T cell-dependent IgA production. Other dendritic subsets (CD8+ cytoid plasma) also induce IL-10 producing Treg cells. The controlling influence of Treg cells can prevent the Th1 response to commensal bacteria which can be responsible for inflammation and tissue damage. Furthermore, the commensal bacteria can attenuate TLR signalling and NF-ï«B activation through inhibition of Iï«B-ï¡ ubiquination, so that NF-ï«B-mediated response can be prevented. Additionally, the mesenteric lymph nodes can limit the mucosal immune induction process to the mucosal immune system itself. (as reviewed by (Blum and Schiffrin, 2003), (Kelly et al., 2004), and (Magalhaes et al., 2007)). In conclusion, the normal gut immune system can interact with commensal bacteria in a beneficial manner, not to kill them but to use them as a trigger factor to modulate the gastrointestinal immune system to be always in a homeostatic situation.
Interactions between the Innate and Adaptive Immune System against Pathogenic Bacteria
The important different feature between commensal and pathogenic bacteria is the presence of virulence factors. Virulence factors determine the pathogenicity of the bacteria and are encoded by organised genes called Pathogenicity Islands. The virulence factors can facilitate bacteria to adhere, to invade, to colonise and to cause infection and disease in a susceptible host. The same virulence factors may not cause infection in an immunised host, so virulence factors may not only be seen independently as pathogenic bacterial characteristic but also as a result of host-pathogen interaction. Generally, the virulence factors can be adherent molecules, invasion systems, enzymes and toxins. Bacteria have different mechanisms which enable to produce toxin in the external environment or to inject the virulence factors directly into the cytosol of the target cell using a secretion system. The secreted virulence factors allow bacteria to cross the epithelial barriers (as reviewed by (Magalhaes et al., 2007)). Several mechanisms can be utilised by bacteria to cross the physical barrier of the epithelium namely invasion of M cells, invasion of epithelial cells or breaking the epithelial lining of the intestine (Janeway, 2005).
The epithelial cells that line the intestine sense the bacteria through the innate immune system. In the gut, there are two classes of pattern recognition receptors (PRRs) called Toll-like receptors and NOD-like receptors which are expressed at the cell surface or associated with intracellular organelles and localise to the cell cytosol, respectively. The host PRRs can distinguish bacterial components which are called pathogen associated molecule patterns (PAMPs) from host components. Twelve different members of TLRs detect different microbial-derived ligands, for example TLR2 detects lipoproteins and lipoteichoic acid, TLR4 detect lipopolysaccharide from Gram negative bacteria, and TLR5 detects flagellin (reviewed in (Kumar et al., 2009) and (Takeuchi and Akira, 2010)). The PAMP recognition by PRR leads to signal delivery to host regarding the presence of infection and trigger proinflammatory and anti microbial response (as reviewed by (Akira et al., 2003) and (Takeuchi and Akira, 2010)).
Following antigen recognition by TLRs, different genes are transcriptionally regulated depending on the TLRs and cell types involved. TLR signalling relies on the usage of the distinct adaptor molecules such as MyD88 and TRIF. Generally, there are two distinct pathways of TLR signalling, namely MyD88-dependent and TRIF-dependent pathways. In the early phase of NF-ï«B and MAPK activation, MyD88-dependent pathway is activated whereas in the late phase, TRIF-dependent pathway is stimulated. Moreover, the latest is also responsible for IRF-3 activation which leads to the induction of IFN-ï¢ and IFN-inducible genes. In the end, TLR signalling contributes to the inflammatory response (reviewed in (Mogensen, 2009), (Kawai and Akira, 2010) and (Takeuchi and Akira, 2010)).
Other pattern recognition receptors are NOD1 and NOD2 which belong to a family of NLRs. These two receptors can serve as intracellular sensors of muramyl dipeptide, a component of peptidoglycan, from bacteria or other pathogens (as reviewed by (Bourhis and Werts, 2007) and (Benko et al., 2008)). The expression of NOD protein is unique. There can be an autocrine stimulation but TLR-induced NF-ï«B activation can lead directly to paracrine stimulation, for example, via IL-8 (Takahashi, 2006, Werts et al., 2007). Thus, NODs synergize with TLRs to induce inflammation in response to pathogens.
Both NODs and TLRs can induce the maturation of human dendritic cells which links between innate and adaptive immune responses. Being activated, the dendritic cells migrate to regional lymph nodes and present the antigenic peptide to relevant MHC molecules. Simultaneously, phagocytosis and upregulation of costimulary molecules happen. Finally, the process switches in chemokine receptor expression and cytokine secretion. Additionally, the stimulated dendritic cells are also able to stimulate naÃ¯ve CD4+ T lymphocytes into different Th subsets, which is, in part, controlled by TLR-induced cytokines (as reviewed by (Mogensen, 2009)). The innate immune response can also initiate further adaptive immune response by inducing Th1 and Th17 response. NOD1 also synergize with TLRs to activate Th1 and Th17 response, which both are important in protection against bacterial infections (as reviewed by (Fritz et al., 2007) and (Mogensen, 2009)). In conclusion, both innate and adaptive immune responses work together to counter the pathogens synergistically.
Pathogenic bacteria differ from non-pathogenic or commensal bacteria due to the presence of specific pathogenicity genes. The genes, which can be organized in pathogenicity islands, are responsible for expression of virulence factors such as secretion system proteins and toxin. Helicobacter pylori and Campylobacter jejuni are pathogens which infect the gastrointestinal mucosa and cause serious sequelae (Lecuit et al., 2004). The pathogenesis and immune response triggered by the pathogens will be discussed briefly in the following sections.
1.2.1. Pathogenesis of Helicobacter pylori Infection and Host Immune Response
Helicobacter pylori infection still presents a major public health problem worldwide. It is estimated that approximately half of the world's population are colonized with H. pylori (Zhang et al., 2009). Although many infected individuals remain largely asymptomatic, H. pylori infection is associated with peptic ulcer disease, gastric ulcers, gastric MALT lymphoma and gastric adenocarcinoma (Uemura et al., 2002, Ferreccio et al., 2007) and as reviewed by (Konturek et al., 2009). Gastric cancer is the fourth most common cancer in the world (as reviewed by (Crew and Neugut, 2006)).
H. pylori infect gastric epithelial cells. The bacteria have several mechanisms to survive in a hostile environment and to colonise the mucosa and interact with the host's epithelial cells to cause chronic inflammation. H. pylori have adapted to live in highly acidic environment of the stomach due to their urease enzyme which can hydrolyse urea into ammonia so that the acidity of the stomach can be neutralised (as reviewed by (Kusters et al., 2006)). Furthermore, H. pylori have several virulence factors which facilitate the bacteria to infect host and elicit an inflammatory response. Certain virulence factors are harboured by certain strains and are associated with specific pathogenesis of the infection. For example, cagPAI genes encode CagA, a protein associated with the increased risk of developing peptic ulcer or gastric cancer. The protein has indirect oncogenic properties and is believed to cause mutation of the tumour suppressor genes (Deguchi et al., 2001) and as reviewed by (Nguyen et al., 2008). Furthermore, cagPAI genes also encode a Type IV Secretion System (TFSS) which can transfer CagA protein and peptidoglycan into host cells and eventually, induce secretion of proinflammatory cytokines including IL-8. The secretion apparatus stimulates intracellular signaling including NF-ï«B and MAPK which leads to the transcription of genes responsible for inflammation and apoptosis (Shibata et al., 2005, Ferrero et al., 2008). Another virulence factor namely vacA, which harboured by 50% of all H. pylori can induce apoptosis and modulate the immune response (as reviewed by (Kusters et al., 2006)).
Both extra cellular and intracellular pattern recognition molecules are important in the recognition of H. pylori. The bacterial component, peptidoglycan, is transferred into the cytoplasm by cag-PAI mediated contact between the epithelial cells and bacterium, and then is recognized by NOD1 (Viala et al., 2004). The peptidoglycan is also recognised by extracellular receptor NOD2 (reviewed in (Kusters et al., 2006)). The role of TLRs in recognition of bacterial components is controversial. Some TLRs such as TLR2, TLR4 and TLR9 can induce NF-ï«B activation and chemokine expression in the gastric cells (Rad et al., 2009), although TLRs do not seem to be predominant in the induction of innate immune responses against H. pylori. TLR5 is less potent in activating immune response because the flagellin of H. pylori is not recognised well by TLR5, so that H. pylori can evade TLR5 immunosurveillance (Gewirtz et al., 2004).
The development of H. pylori disease is determined by many factors regarding the host-pathogen relationship during the infection. Both innate and adaptive immune responses are of importance to eradicate the pathogen, but most individuals get chronic infection probably because the immune system does not clear the infection. The failure of one of the component can lead to the progression of the disease. Naturally, the innate immune response take few days after pathogen invasion then is followed by initiation of adaptive immune response. H. pylori are able to stimulate maturation of dendritic cells which leads to the stimulation of Th1 cells, which is TLR-dependent (Algood et al., 2007, Rad et al., 2007).
Another innate immune component which has an important role in the inflammation process caused by H. pylori is mucin. This molecule is of interest to be investigated regarding the fact that mucin is the first molecule that bacteria have to cope with during penetration to gastric epithelial cells. A very recent study very relevant to my project has showed that MUC1 can attenuate epithelial inflammation in response to H. pylori infection. This attenuation is caused by inhibition of Iï«Bï¡ phosphorylation, NF-ï«B inactivation which finally prevent further inflammation (Guang et al., 2010). However, the crosstalk between MUC1 and TLR and NOD signalling in modulating epithelial immune response needs to be investigated further.
1.2.2. Pathogenesis of Campylobacter jejuni Infection and Host Immune Response
Campylobacter jejuni is a major cause of diarrhoeal disease in humans and is generally regarded as one of the most common bacterial causes of gastro-enterocolitis in many countries including Australia. The campylobacteriosis outbreak in Australia during 2001 to 2006 showed that contaminated food and poultry is the important vehicles to transmit the disease (Unicomb et al., 2009). The high incidence of diarrhoea caused by Campylobacter jejuni and its possible sequelae, makes this infection highly important in terms of socio-economic impact (Ivanova, 2010). Additionally, Campylobacter jejuni infection can cause post-infection complications such as MALT lymphoma(Schmidt-Ott et al., 2006) (Lecuit et al., 2004) and Guillain-Barre syndrome (Schmidt-Ott, 2006).
C. jejuni infects the small and large intestine. After ingestion, the pathogenesis of C. jejuni infection is commenced when the bacteria penetrate the intestinal mucus layer and the production of bacterial protein toxins called cytolethal distending toxin (CDT). It has been shown that C. jejuni utilize outer membrane vesicles to transport all the sub unit of cytolethal distending toxin. The cell cycles can extremely be arrested by the toxin resulting in cell death (Lindmark et al., 2009). The release of the toxin then can cause epithelial damage because of the toxin itself and because of the inflammatory response to the bacteria. The subsequent infection can then trigger host's immune response both mucosal and systemic antigen-specific IgA and IgG both in human and in ferret model (Cawthraw et al., 2002, Nemelka et al., 2009).
However, C. jejuni has evolved mechanism to escape from host's innate immune system by evading from TLR 5- and TLR 9-mediated immunosurveillance. Normally, flagellin, the structural component of flagella, can trigger intestinal epithelial cells to express proinflammatory genes but C. jejuni have amino acids changes in their flagellin at the TLR5 recognition site. Therefore, C. jejuni flagellin is a poor agonist of TLR to induce proinflammatory cytokine production (Andersen-Nissen et al., 2005, Watson and Galan, 2005). Such immune evasion is aimed to make the infection persist. However, upon C. jejuni infection, the bacterial component can be recognised by host internal pattern recognition receptor namely NOD1 and then NF-ï«B is activated leading to IL-8 production (Johanesen and Dwinell, 2006, Zilbauer et al., 2007). Thus, C. jejuni can induce IL-8 production independently of TLR5 activation. It has been studied that C. jejuni infection can induce IL-8 secretion due to bacterial invasion and the toxin/CDT (Hickey et al., 1999, Hickey et al., 2000). The signalling pathway responsible for IL-8 production is Mitogen-activated Protein Kinase including extracellular signal-regulated kinase (ERK) and p38 MAP kinase with the ERK pathway being vital for IL-8 induction (Watson and Galan, 2005).
The first step of pathogenesis in C.jejuni infection is the penetration of intestinal mucosal layer by C. jejuni. It is a critical step to ensure the further invasion by the pathogen. The presence of mucin in the epithelial surface has a great benefit to prevent further damage triggered by the pathogen. It was shown that mucin (MUC1) can protect the epithelial cells from the effects of cytolethal distending toxin and limit the C. jejuni infection (McAuley et al., 2007). Another epithelial surface mucin is MUC13 which is expressed in gastrointestinal epithelial cells. Although MUC13 is the same family as MUC1 (cell surface mucin), they may have differences in the modulation of immune responses as regards intracellular signalling.
Mucins are Part of The Immune System
The Characteristics of Mucins
The lining of entire gastrointestinal tract is protected by a mucus layer. The main components of the mucus layer are mucin glycoproteins which are produced by mucus-producing cells in the epithelium or submucosal glands. The physical and biochemical characteristic of the mucin is closely related to their function, for example, high viscoelasticity is necessary for protection from mechanical damage and as a trap for microbes. The viscoelasticity is produced by the high molecular weight of mucin macro-molecular complexes and the high hydrated nature of mucus (as reviewed by (Linden et al., 2008b) and (Byrd and Bresalier, 2000)).
Mucins are large glycoproteins with O-linked oligosaccharides which are the main component comprising over 70% of the total mass. The O-glycosylated glycoprotein state will influence the biochemical properties and the function of mucin. The types of carbohydrate structure on mucin or glycosylation depends on tissue-specific enzyme expression, host and environmental factors such as can be altered during bacterial infection or in the presence of mucosal disease (as reviewed by (Linden et al., 2008b) and (Dharmani et al., 2009))
The expression and distribution of mucins in different tissues are determined by 21 different genes. The different type of mucin is dependent on the structure and location which can be subdivided into secretory and cell-surface mucin (reviewed in (Dharmani et al., 2009)). The following discussion is mainly about the cell surface mucins which will be studied in my research.
The Cell Surface Mucins
Cell surface or membrane bound mucins are transmembrane glycoproteins that belong to family comprising MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17 and MUC20. Among them, MUC1 and MUC4 are well-characterised. Membrane bound mucins are present on the apical membrane of all mucosal epithelial cells and contain large extracellular domains predicted to form rigid elongated structures. With respect to their high expression, these molecules are probably to be a dominant constituent of the glycolayx (as reviewed by (Linden et al., 2008a) and (Jonckheere and Van Seuningen, 2009)).
Cell surface mucins have two domains namely the extracellular and cytoplasmic domains. The extracellular domain forms an extremely large thread-like structure covered by complex of O-linked oligosaccharide and can be shed from the cell surface. The cytoplasmic domains of the individual mucins are highly conserved across species and have an important role in signal transduction due to the ability to interact with kinases and adaptor molecules and undergo tyrosine phosphorylation (Meerzaman et al., 2001, Thompson et al., 2006). The MUC1 cytoplasmic domain can be cleaved and then the cleaved domain translocates to mitochondria and together with p53 transcription factor, translocate to the nucleus to modulate cell cycles and prevent apoptosis caused by genotoxic stress (as reviewed by (Linden et al., 2008b)). Most cell-surface mucins appear to be cleaved into two sub units during synthesis. The cleavage process occurs via auto proteolysis. Additionally, the extracellular ï¡-subunit can be shed from the cell surface either mediated via a second distinct cleavage event or by physical shear (as reviewed by (Linden et al., 2008a)).
MUC1 is expressed by most mucosal tissues including stomach and intestinal tissue. MUC1 has been estimated to be 200-500 nm in length, so that it will exceed other molecules attached to the plasma membrane. The molecule surface contains complex array of O-linked oligosaccharides that have been shown to bind to microbial molecules. For example, distinct set of carbohydrates play an important role in adhesion of H. pylori to epithelial cells, attenuation of H. pylori colonisation and facilitation of inflammatory response following H. pylori infection (as reviewed by (Linden et al., 2008b) and (Kobayashi et al., 2009)).
Much research has been done to investigate the regulation of mucin secretion and their role in signalling pathways both in normal cells and malignant cells. MUC1 is produced by most normal epithelial cells but MUC1 is also produced by mammary, pancreatic and colon cancer cells. The different mucin expression in normal, preneoplastic and neoplastic cells is determined by different genes leading to different phenotypes such as the O-linked carbohydrates on MUC1 mucin differ between malignant and normal epithelial cells. Such difference can be detected using different monoclonal antibodies (reviewed in (Ho et al., 1995) and (Byrd and Bresalier, 2000)).
Regarding the role of MUC1 in signalling pathways, the purpose appears to be to protect the epithelial cells from further damage and enhance cell survival. MUC1 can modulate signalling pathway in response to oxidative stress in cancer cells. Additionally, MUC1 is also involved in NF-ï«B signalling (Yin et al., 2004, Ahmad et al., 2007). Consequently, the activated NF-ï«B pathway will activate transcription of proinflammatory and anti-apoptotic genes (reviewed in (Thompson et al., 2006)).
MUC13 is highly expressed in the gastrointestinal tract which has slightly different gastrointestinal expression pattern from MUC1 as in stomach; MUC1 is more expressed than MUC13; whereas in intestine, MUC13 is more dominant than MUC1. Like MUC1, MUC13 has similar structure comprising extracellular subunit and cytoplasmic domain. MUC13 is also expressed in trachea, kidney, appendix, middle ear epithelium and hemopoietic cells ((Williams et al., 2001) and as reviewed by (Linden et al., 2008b)). MUC13 is also expressed in malignant cells such as colorectal, oesophageal, gastric, pancreatic and lung cancer. It has been studied that MUC13 has an important role in the development of ovarian cancer due to involvement in signalling pathway (Chauhan et al., 2009).
The Role of Mucins against Pathogenic Bacteria
With regard to pathogenic bacteria, there are important interactions between mucins and bacteria. These interactions will determine the fate of the microbes and the diseases they cause. The structure and physical property of mucins allow these molecules to form a physical barrier to pathogen invasion. For example, MUC1 can shield the epithelial surface from exposure to H. pylori virulence factors, preventing adhesion-dependent synthesis of proinflammatory cytokines. However, the carbohydrate structure of mucins favour bacteria to colonise and to use it as an energy source and eventually bacteria can exploit underlying signalling pathways. Such stimuli will typically enhance the mucus secretion in response to bacteria (reviewed in (Byrd and Bresalier, 2000) and (Dharmani et al., 2009)).
The secretion of mucin can be altered during infection and inflammation where pathogenic bacteria can use various pathways to induce or reduce mucin secretion. LPS from H. pylori can activate caspase-3 and induce apoptosis which is dependent on p38 MAPK. It was shown that LPS of H. pylori can inhibit mucin glycosylation which may have deleterious effects on mucin assembly. The decreased mucin synthesis further increases H. pylori adhesion leading to disease (reviewed in (Byrd and Bresalier, 2000) and (Dharmani et al., 2009)).
However, McGuckin et al has shown that MUC1 can prevent H. pylori colonisation due to the function of MUC1 as a "releasable decoy ligand". The extracellular domain can be cleaved by host protease and subsequently shed the bacteria from epithelial surface (McGuckin et al., 2007). This result is also supported by McAuley who found that MUC1 also can also limit the attachment of C. jejuni to epithelial surface and prevent further invasion. Moreover, following release of the extracellular domain, the cytoplasmic domain together with p53 play an important role in cell signalling related to the modulation of cell growth and apoptosis in response to bacterial toxin (McAuley et al., 2007). In conclusion, the presence of MUC1 is important to prevent the more severe pathology following bacterial infection.
As mentioned earlier that bacterial infection is a trigger for proinflammatory responses. The main purpose of inflammation in the context of bacterial infection is to resolve the infection rapidly and efficiently. However, the inflammation reaction may cause tissue damage. Therefore, the host's immune system needs a counter mechanism to avoid excessive tissue damage but still effectively neutralise danger effects caused by pathogens. Such mechanism will help to prevent further inflammation and to reduce the severity of the pathologic effects. Mucin glycoproteins have been shown to play an important role to prevent the development of chronic inflammation. It was shown by Ueno et al (Ueno et al., 2008) that the reduced proinflammatory response in cells bearing mucin was most likely due to the suppression of TLR-mediated NF-ï«B activation by MUC1. In a very recent study conducted by Guang et al also showed that MUC1 can attenuate epithelial inflammation in response to H. pylori infection. Such attenuation is performed by suppressing the NF-ï«B pathway via inhibition of Iï«Bï¡ phosphorylation (Guang et al., 2010). Therefore, mucins may be crucially important to prevent the development of chronic inflammation.
In this proposed study, crosstalk between mucin and pattern recognition molecules will be investigated to define the mechanism of epithelial immune response modulation by mucin against pathogens to prevent further inflammation, and will focus on the MUC1 and MUC13 cell surface mucins.
It is hypothesized that cell surface mucins (MUC1 and MUC13) modulate inflammatory signalling by gastrointestinal epithelial cells in response to pathogenic bacteria.
The project aims to characterise the response of gastrointestinal epithelial cells to:
live pathogenic bacteria,
virulence factors of pathogenic bacteria
TLR- and NOD-ligands,
in the presence and absence of MUC1 or MUC13.
To this end, the MUC1 and MUC13 genes will be knocked down in gastric and intestinal cell lines using siRNA transfection as well as primary gastric and intestinal cells will be isolated from Muc1-/-and Muc13-/-knockout mice, respectively.
The principle of the experiment is co-culture gastrointestinal epithelial cells with the pathogens or pathogen-derived substances that can induce proinflammatory cytokine production. With respect to the outcome of this study namely comparison of the inflammatory response in the presence and absence of MUC1 and MUC13, the gastrointestinal cells which express MUC1 or MUC13 and those cells which do not express MUC1 or MUC13 will be used. Then, NF-ï«B activation transcription factor binding assays on nuclear extracts and IL-8 ELISA assays on tissue culture supernatants will be performed to characterise inflammatory signalling by co-cultured epithelial cells.
The proposed study of MUC1 is substantially different from the very recently published study by Guang et al (Guang et al., 2010). The gastric cell lines will be used in this study is MKN7 cells which has an ability to adhere in a continuous polarized layer. This characteristic of the cell lines is very important to ensure bacteria to attach to the apical surface of the epithelial cells (Linden et al., 2007).
To achieve each aim, the study will be conducted as follows:
To characterise the immune response modulation of gastrointestinal cells to pathogenic bacteria by MUC1 and MUC13
Gastric model : primary gastric epithelial cell from Muc1-/- knockout mice and Muc1+/+ wild type mice, MKN7 human gastric epithelial cells with high MUC1 expression and MKN7 Muc1 si RNA transfectant; Helicobacter. pylori strain J99;
Intestinal model: primary intestinal epithelial cell from Muc13-/- knockout mice and Muc13+/+ wild type mice. LS513 human intestinal epithelial cells with high MUC13 expression and LS513 MUC13 si RNA transfectant; Campylobacter jejuni strain 81116
Experiment: co-cultures of bacteria and mammalian epithelial cells with or without mucin expression will be established over time courses 4, 8, 12 and 24 hour with the same number of bacteria. Micro-aerobic co-culture conditions are used in this study.
Readout: NF-ï«B activation (commercial transcription factor assay and inflammatory cytokine production (IL-8) using ELISA
To characterise the immune response modulation of gastrointestinal cells to bacterial toxin by MUC1 and MUC13
If the co-cultures of bacteria and mammalian cells give the significant different result (readout), the experiment will be continued further to investigate which part of the bacteria that can induce inflammatory response
Gastric model: cells and culture condition as for aim 1. The bacterial virulence factors (toxin) will be obtained from Helicobacter pylori J99 lysates and purified CagA and VacA toxin
Intestinal model: cells and culture condition as for aim 1. The bacterial virulence factors (toxin) will be obtained from Campylobacter jejuni 81116 lysates and purified CDT.
Experiment: co-cultures of bacterial lysates and mammalian epithelial cells with or without mucin expression will be established over time courses 4, 8, 12 and 24 hour as for Aim 1.
Readout: as for Aim 1.
To characterise the immune response modulation of gastrointestinal cells to TLR- and NOD1- and NOD2-ligands
The expression of TLRs and NODs on mammalian epithelial cells needs to be investigated first before performing co-culture of mammalian cells with relevant TLR- and NOD-ligand.
Gastric model: both murine primary gastric cell and cell lines as for Aim 1.
Intestinal model: both murine primary intestinal cell and cell lines as for Aim 1
Experiment: co-cultures of mammalian cells with ligands for relevant TLR, such as Pam3Cys lipopeptide (for TLR2), LPS (TLR4), flagellin (TLR5), CpG DNA (TLR9), peptidoglycan (ligand for NOD1) and muramyl dipeptide (NOD2), with the culture condition as for Aim 1
Readout: as for Aim 1.
Statistical analysis: to analyse the significant difference in immune response in the presence and absence of MUC1 and MUC13, the results will be analysed using Mann Whitney test.
Techniques that are going to be used in this experiment:
Co-culture of epithelial cells and bacteria
Real time PCR, to study the level of expression of MUC1 and MUC13 in gastric and intestinal epithelial cells, respectively.