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Mechanisms of enteroaggregative escherichia coli flagellin-induced IL-8 secretion from epithelial cells Khan, Mohammed Aatif Shah 2003

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Mechanisms of Enteroaggregative Escherichia coli flagellin-induced IL-8 secretion from epithelial cells by Mohammed Aatif Shah Khan, M D Spartan Health Sciences University, St. Lucia, West Indies. 1988. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies Department of Experimental Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF'BRITISH C O L U M B I A / June 2003 © Mohammed Aatif Shah Khan In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea in a number of clinical settings. E A E C diarrhea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. The molecular pathogenesis of E A E C diarrhea is not understood completely. Flagellin (FliC-EAEC), a major bacterial surface protein of E A E C , has been shown to induce IL-8 release from several epithelial cell lines. The aim of this study is to elucidate the mechanisms of IL-8 secretion by Fl iC-EAEC in epithelial cells lines. Toll-like receptors (TLRs) have been described as important component of the innate immune response in humans. It is known that host response to Gram-negative flagellin is mediated by TLR5, which signals through N F - K B to induce transcription of pro-inflammatory cytokines. Our results show that Fl iC-EAEC activates Akt (PKB) and 1-KB degradation (downstream targets of PI-3K) as observed in Western blots. Experiments with pharmacological inhibitors and dominant negative PI-3K indicated that PI-3K/Akt signaling is not necessary for Fl iC-EAEC induced IL-8 secretion. p38 MAP kinase is one of a family of stress-related kinases that influence a diverse range of cellular functions including inflammatory responses to microbial products. To determine whether this enzyme mediates IL-8 release, a pharmacological inhibitor of p38 MAP kinase, SB 203580, was used to inhibit Fl iC-EAEC induced IL-8 release. We found that IL-8 secretion is regulated by p38 MAP kinase, which undergoes activation in Caco-2 and THP-1 cells, as determined by Western analysis. Exposure of HEp-2 cells transiently expressing TLR5 resulted in p38 MAP kinase dependent IL-8 secretion. Activation of IRAK was also observed in TLR5-transfected HEp-2 cells after treatment with FliC-EAEC. These results suggest that Fl iC-EAEC-induced IL-8 secretion is regulated by p38 MAP kinase and involves TLR5 in epithelial cells. ii Table of Contents Abstract ii Table of Contents i i i List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xii Chapter I: Introduction 1 1.1 The epidemiology and history of infectious diarrhea 1 1.2 Vaccines and antibiotic resistance 2 1.3 Etiology of bacterial diarrheas 3 1.4 Diarrheagenic Escherichia coli , 4 1.5 Pathogenesis of Enteroaggregative Escherichia coli (EAEC) diarrhea 8 1.5.1 EAEC adherence and virulence factors 11 1.6 The Innate immune system 14 1.6.1 Toll-like receptors 15 1.6.2 TLR ligands 16 1.7 Toll-like receptor (TLR) signaling pathways 18 1.8 Mitogen-activatedprotein kinase signaling pathway 21 1.8.1 Transcription factors regulated by p38 MAP kinase 22 1.9 Regulation of interleukin-8 (IL-8) transcriptional activation 24 1.10 Biological functions of IL-8 26 i i i 1.11 Mechanisms of inflammation in human intestinal epithelium 28 1.12 Toll-like receptors (TLR) and intestinal epithelium 30 1.13 The relevance of TLR signaling pathways 32 1.14 Hypothesis and objectives 33 1.15 Caco-2 intestinal epithelial cells: a model for studying intestinal inflammation 35 1.16 THP-1 promonocytic cell line: a model for studying host-pathogen interaction 36 1.17 Human epithelial HEp-2 cell line: a model for studying host immune responses 38 Chapter II: Materials and Methods 41 2.1 Caco-2 cell culture for studies of PE3K and p38 MAP kinase activation by FliC-EAEC 41 2.2 THP-1 cell culture for studies of IL-8 secretion, PI-3K and p38 MAP kinase activation 41 2.3 TLR5 transient expression in HEp-2 cells 41 2.4 IL-8 secretion from U937promonocytic cell line incubated with FliC-EAEC 42 2.5 Expression and purification of EAEC flagellin 43 2.6 Enzyme-linked immunosorbent assay (ELISA) for measurement of IL-8 secretion 46 2.7 Pharmacological inhibitors of signaling proteins 48 2.8 Western blotting and immunoprecipitation 48 iv 2.9 The determination of total cell lysate protein concentration 49 2.10 Polyacrylamide gel electrophoresis of cellular proteins 49 2.11 Western blotting for detection of cellular proteins by chemiluminescence 50 2.12 Generation of a mutant TLR5 51 2.13 In-vitro enzyme assay for measurement of PI-3K activity 53 Chapter III: Results 54 3.1 FliC-EAEC activates Akt and degrades I-KB in Caco-2 cells 54 3.2 FliC-EAEC activatesp38 MAP kinase in Caco-2 cells 63 3.3 FliC-EAEC causes IL-8 secretion, Akt andp38 MAP kinase activation in THP-1 cells 70 3.4 FliC-EAEC treatment ofHEp-2 cells transfected with TLR5 causes p38 MAP kinase-dependent IL-8 secretion and IRAK activation 71 Chapter IV: Discussion 86 4.1 Effects of FliC-EAEC on PI-3K signaling pathways in Caco-2 cells 86 4.2 Activation ofp38 MAP kinase by FliC-EAEC in Caco-2 cells 88 4.3 Activation of Akt and p38 MAP kinase by FliC-EAEC in THP-1 cells 92 4.4 Activation of p38 MAP kinase and IRAK in TLR5 transfected HEp-2 cells 93 Chapter V: Conclusions and future directions 97 5.1 PE3K/Akt signaling by FliC-EAEC 97 5.2 Regulation of FliC-EAEC-inducedIL-8 secretion by p38 MAP kinase activation 99 5.3 FliC-EAEC activates TLR5 signaling pathways in epithelial cells 100 5.4 Implications of FliC-EAEC signaling and EAEC diarrhea 101 Bibliography 104 vi List of tables Table 1: E A E C virulence factors vii List of Figures Figure 1: A n outline of the mechanisms of bacterial infection in human epithelium 7 • Figure 2: Stacked brick configuration of E A E C 9 Figure 3: Proposed Toll-like receptor 5 signaling pathways 19 Figure 4: Purified F l iC-EAEC 45 Figure 5: F l iC-EAEC activation of PI-3K in Caco-2 cells 55 Figure 6: FliC-EAEC-induced PI-3K tyrosine phospho-activation 56 Figure 7: Phospho-activation of Akt by Fl iC-EAEC in Caco-2 cells 58 Figure 8: 1-KBOC degradation by Fl iC-EAEC in Caco-2 cells 59 Figure 9: IL-8 secretion from WT and D N PI-3K U937 cells 61 Figure 10: Effect of N F - K B and PI-3K inhibition on F l iC-EAEC induced IL-8 release from Caco-2 cell 62 Figure 11: Effect of p38 M A P kinase and N F - K B inhibition on F l iC-EAEC- induced IL-8 release from Caco-2 cells 65 Figure 12: Effect of p38 M A P kinase inhibition on IL-8 secretion from Caco-2 cells incubated with F l iC-EAEC 66 Figure 13: Phospho-activation of p38 M A P kinase by F l iC-EAEC in Caco-2 cells 67 Figure 14: Time-course of p38 M A P kinase phospho-activation by F l iC-EAEC 68 Figure 15: Inhibition of FliC-EAEC-induced p38 M A P kinase phospho-activation 69 Figure 16: Time-course of FliC-EAEC-induced IL-8 secretion from THP-1 cells 72 Figure 17: Dose-response of FliC-EAEC-induced IL-8 secretion from THP-1 cells 73 Figure 18: Inhibition of IL-8 secretion from THP-1 cells exposed to F l iC-EAEC 74 viii Figure 19: Activation of Akt by Fl iC-EAEC in THP-1 cells 75 Figure 20: F l iC-EAEC activates p38 M A P kinase in THP-1 cells 76 Figure 21: F l iC-EAEC induction of IL-8 secretion from HEp-2 cells transiently transfected with TLR5 79 Figure 22: Inhibition of FliC-EAEC-induced IL-8 secretion from TLR5 transfected HEp-2 cells 80 Figure 23: Activation of p38 M A P kinase in TLR5-transfected HEp-2 cells exposed to F l iC-EAEC 81 Figure 24: F l iC-EAEC induction of IL-8 release from HEp-2 cells transfected with wild type and Y798L TLR5 82 Figure 25: Activation of p38 M A P kinase by Fl iC-EAEC in HEp-2 cells transfected with wild type and Y798L TLR5 83 Figure 26: Activation of IRAK by Fl iC-EAEC in Caco-2 cells 84 Figure 27: IRAK activation in TLR5-transfected HEp-2 cells treated with F l iC-EAEC 85 Figure 28: A proposed model of FliC-EAEC-induced IL-8 signaling pathways in epithelial cells 103 ix List of abbreviations E A E C Enteroaggregative Escherichia coli ETEC Enterotoxigenic Escherichia coli EHEC Enterohemorrhagic Escherichia coli EPEC Enteropathogenic Escherichia coli EIEC Enteroinvasive Escherichia coli STEC Shiga-toxin producing Escherichia coli D A E C Diffusely adherent Escherichia coli IL-8 Interleukin-8 cAMP cyclic adenosine monophosphate I K K Inhibitory kappa-B kinase I-KB Inhibitory kappa-B N F - K B Nuclear factor kappa-B NF-IL-6 Nuclear factor-IL-6 PI-3K Phosphatidylinositol 3-kinase Akt Protein kinase B (a target of PI-3K) M A P K Mitogen activated protein kinase E R K Extracellular regulated protein kinase JNK c-Jun NH2-terminal kinase A A F Aggregative adherence fimbriae Pet Plasmid-encoded toxin EAST Enteroaggregative heat stable toxin STa Escherichia coli heat-stable enterotoxin LPS Lipopolysaccharide L A M Lipoarabinomannan A / E Attaching and effacing P A M P Pathogen associated molecular pattern TIR Toll-interleukin 1 receptor LPB Lipopolysaccharide binding protein IFNy Interferon gamma TNFa Tumor necrosis factor alpha GMCSF Granulocyte macrophage colony stimulating factor MCP-1 Monocyte chemotactic protein-1 RANTES Regulated upon activation normal T cell expressed and presumably secreted IGF-II Insulin-like growth factor-II Thl T helper cells of Thl subtype CD14 Cluster designation 14 receptor PRR Pattern recognition receptor L R R Leucine-rich repeat TLR Toll-like receptor MD2 A receptor required for TLR4 signaling IRAK Interleukin-1 receptor-associated kinase MyD 88 ^ Myeloid differentiation factor 88 IL-1R Interleukin-1 receptor TRAF-6 Tumor necrosis factor receptor-associated factor-6 X C 0 X 2 Cyclooxygenase-2 iNOS Inducible nitric oxide synthase TIRAP Toll/Interleukin-1 receptor domain containing adaptor protein TOLLIP Toll-like receptor interacting protein M A L MyD88 adaptor-like TGFP Transforming growth factor beta T A B TGFp-activated protein kinase 2-binding protein T A K TGFp-activated kinase M A P K Mitogen-activated protein kinase M E K MAPK/extracellular-signal-regulated kinase (ERK)-kinase M K K K Mitogen-activated protein kinase kinase kinase SAPK Stress-activated protein kinase H0G1 gene encoding M A P kinase M A P K A P MAPK-activated protein kinase-activated protein kinase Max transcription factor interacting with Myc Myc transcription factor targeted by p38 M A P kinase AP-1 Activator protein-1 ATF Activating transcription factor-1 bZIP Basic leucine zipper transcription factors e.g. c-Jun TRE 12-0-tetradecanoylphorbol-13 acetate-TP A responsive element CRE cAMP responsive element CREB cAMP responsive element-binding protein C/EBP CAAT/enhancer- binding protein A R E Adenylate/uridylate-rich elements in messenger R N A D N A Deoxyribonucleic acid R N A Ribonucleic acid RT-PCR Reverse transcriptase polymerase chain reaction BCL-2 A family of pro-survival proteins H L A Human leukocyte antigen IEC Intestinal epithelial cells HSP Heat shock protein GFP Green fluorescent protein FBS Fetal bovine serum PBS Phosphate-buffered saline ELISA Enzyme-linked immunosorbent assay SDS-PAGE Sodium dodecylsulphate-polyacrylamide gel electrophoresis P B M C Peripheral blood mononuclear cells MIC Macrophage inhibitory protein xi Acknowledgements I would like to acknowledge the contributions of others who have helped in completion of this research project. First and foremost, I am grateful to my supervisor, Dr. Ted Steiner, for his guidance and supervision throughout this project. He was always attentive to my problems and offer practical advice, which benefited my experiments. In addition, he has been very kind and patient with me, which allowed me to improve the standard of my work in the laboratory. I have learnt many things from him and will try to inculcate these qualities in future. This work would not have been possible without the cooperation of Jian Kang and Bemulu Baraki, who have assisted in making my experiments successful in the laboratory. I would like to mention Dr. Zakaria Hmama, who has been a mentor and a friend. He has taught me to critically analyze my work and optimize my experimental protocols. Members of his laboratory have assisted with experiments and also supported me at a personal level. My graduate committee has provided direction and focus in my work. Their suggestions and comments enabled me to make significant progress at various stages of this project. I am also grateful to colleagues in department of Experimental Medicine who have been good friends at times of need and encouraged me throughout my studies. Finally, I am thankful to department of Experimental Medicine and UBC for giving me an opportunity to learn science. xii Chapter I: Introduction 1.1 The epidemiology and history of infectious diarrhea Diarrhea is a common illness that patients present with to their family doctors. Infectious diarrhea affects all age groups in developing regions of the world, whereas in developed countries, children in day care, elderly in nursing homes and travelers are affected more frequently. Excessive use of antibiotics, malnourishment and impairment of the immune system contribute to the increasing disease burden due to infectious diarrhea worldwide. By far, children in the developing world bear the biggest disease burden, making it the leading cause of potential years of life lost [1]. The risk of diarrhea is greatest when children are weaned from breastfeeding, and increases with introduction of food prepared in unhygienic manner. Debilitated patients at the extremes of age have a greater risk of dying from severe diarrhea, usually because of dehydration. In 1982, in a review of morbidity and mortality rates due to diarrhea globally from 1955 to 1979, Snyder and Merson estimated that approximately 4.2 million deaths per year occurred from diarrhea, mostly in young children [2]. In this group, the diarrhea-specific annual mortality rate was 14 per thousand in children < 5 years of age and 23 per 1000 in children < 1 year of age [2]. In an update in 1992, Bern and colleagues summarized studies published from 1980-1990 and observed a decrease in overall global mortality rate to approximately 3.3 million per year [3]. However, a disturbing finding was an increase in the morbidity rate. Guerrant and colleagues noted a similar trend recently that diarrheal disease mortality declined but the morbidity increased worldwide [4]. Much of the success in the decline of mortality rate due to diarrhea is attributable to increased use of oral rehydration therapy (ORT). Unfortunately, ORT does not prevent 1 the onset of diarrhea and while it continues to play a very important role in decreasing mortality, therapeutic strategies to decrease morbidity due to diarrheal infection have not been successful. 1.2 Vaccines and antibiotic resistance Vaccines against diarrheal pathogens have had limited success, mainly due to the diverse nature of the pathogenesis of bacterial infection and incomplete understanding of the molecular mechanisms of host-microbe interaction. Vaccines for bacterial diarrheas such as typhoid fever and cholera are currently being used clinically. Two typhoid vaccines have been recently developed. Ty21a, an attenuated strain of S. typhi administered orally and Vi , the purified bacterial capsule have appeared less toxic than the older whole cell vaccines and are equally effective [5]. In the United States, a parenterally administered phenol-inactivated cholera vaccine is available, but this is poorly tolerated and has limited efficacy. In other countries, two additional cholera vaccines are commercially available: an oral killed whole cell-cholera toxin recombinant B subunit vaccine (WC-rBS) and an oral live attenuated Vibrio cholerae vaccine (CVD 103-HgR). These oral vaccines are well tolerated. In field trials, WC-rBS provides 80%-85% protection from V. cholerae serogroup 01 for at least 6 months. In volunteer studies, C V D 103-HgR provides 62%-100% protection against V. cholerae for at least 6 months. Unfortunately, no commercially available cholera vaccine protects against disease caused by V. cholerae serogroup 0139 [6]. While new vaccines are being developed, a vaccine against common diarrheal pathogens would have a huge impact on child survival and decrease morbidity in the developing regions of the world. Presently, there are no vaccines against any strains of diarrheagenic Escherichia 2 coli in clinical use. The development of a vaccine against emerging Enteroaggregative Escherichia coli (EAEC), which causes persistent diarrhea in children and some travelers, has been hampered by antigenic heterogeneity of E A E C strains. The best approach to E A E C vaccine development would be to identify a highly conserved, highly prevalent, surface- exposed immunogenic protein that could be expressed in an attenuated vector vaccine. Until such candidate vaccines are identified and tested, the disease burden due to E A E C diarrhea, especially in the developing countries is likely to remain a major problem. 1.3 Etiology of bacterial diarrheas Common pathogenic strains of bacteria causing diarrhea worldwide include Salmonella (enteric fever and gastroenteritis), Shigella (bacillary dysentery), Campylobacter jejuni, Escherichia coli and organisms that cause toxin-mediated food poisoning such as Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. The term 'gastroenteritis' is often used interchangeably with infectious diarrhea and usually implies infection of the gastrointestinal tract (small and/or large intestine). Almost all agents causing diarrhea can be acquired from ingestion of contaminated foods or water. Infection by person-to-person contact also occurs and inanimate objects may play a role in the spread of the disease. In each of these settings, the contact facilitates fecal-oral spread of the organism. Meticulous attention to foods and beverages that an individual consumes can significantly decrease the risk of infectious diarrhea. Precautions such as drinking chemically treated water, filtered water, or boiled water and avoiding unpasteurized milk reduces the chances of bacterial diarrhea [7]. Some foods and beverages are particularly associated with specific organisms. 3 Water has been a well-documented vehicle for outbreaks of cholera (V. cholerae), dysentery (amebiasis or shigellosis) and typhoid fever in developing countries. In North America, Giardia and Cryptosporidium are the most frequently reported waterborne infections causing diarrhea. A variety of pathotypes of E.coli cause diarrhea that is transmitted by drinking contaminated water. In addition, eating improperly cooked contaminated foods, particularly in the developed countries where adequate treatment of public water supply and sewage disposal has reduced the incidence of water-borne bacterial diarrhea, may transmit diarrheagenic E. coli [8]. 1.4 Diarrheagenic Escherichia coli Escherichia coli (E. coli) are a member of the genus Escherichia within the family Enterobacteriaceae and the tribe Escherichia, and consists of mostly motile, gram-negative bacilli [9]. When Theodore Escherich first described E. coli in 1884 as an enteric pathogen, he could not have envisioned the complexities of this facultative organism. Escherichia coli colonizes the gut of newborns soon after birth and thereafter continues to be present in the intestine of humans functioning as a commensal [10]. E. coli was recognized as a pathogen causing diarrhea with the finding in 1898 that serum from diarrhea patients agglutinated isolates of E. coli from other patients in the same outbreak but not those of controls [8]. Commensal E. coli is usually harmless and remains confined to the intestinal lumen. However, in immunocompromised hosts or when gastrointestinal barriers are disrupted, even otherwise non-pathogenic strains of E. coli can cause infection [8]. Before the virulence factors of E. coli were identified, pathogenic strains were distinguished mainly by serotyping. A scheme was proposed by Kauffman in 1944 by 4 which E. coli were serotyped on the basis of their O (somatic), H (flagellar) and K (capsular) surface antigen profiles [9]. A total of 170 different O antigens, each defining a serogroup, are recognized presently. A specific combination of O and H antigens defines the serotype of an isolate [8]. To identify a diarrheagenic strain of E. coli, the organism must be differentiated from nonpathogenic gut flora. To a limited extent, serotypic markers correlate with specific categories of diarrheagenic E. coli. However, these markers are generally not reliable by themselves to accurately identify a strain as diarrheagenic (an exception is the serotype 0157:H7, which serves as a marker for virulent enterohemorrhagic E. coli). Serotyping has limited sensitivity and specificity, is tedious, and is performed reliably only in a small number of reference laboratories. Therefore, the detection of diarrheagenic E. coli has become increasingly dependent on identification of virulence characteristics of these organisms. This may include in vitro phenotypic assays, which correlate with the presence of specific virulence traits such as adherence, toxins, or the detection of genes encoding these traits. One of the most useful phenotypic assays for the diagnosis of diarrheagenic E. coli is the HEp-2 assay. First described by Cravioto et al in 1979 [11], this assay remains the "gold standard" for the diagnosis of enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC). In this assay, bacteria are observed for adherence to HEp-2 cells in the presence of D-mannose (to inhibit type I fimbrial adhesins). Currently, there are three well-recognized, distinct patterns of adherence that characterize three classes of diarrheagenic E. coli: E A E C , D A E C , and enteropathogenic E. coli (EPEC). Other adherence patterns, such as chain-like adherence, have been reported, although extensive clinical correlations are lacking [12, 13]. 5 Diarrheagenic E. coli strains were among the first pathogens for which molecular diagnostic methods were developed. These methods are the most reliable and popular for distinguishing certain diarrheagenic categories of E. coli from nonpathogenic isolates in stool samples. DNA probes have been developed for detection of heat labile (LT) and heat stable (STa) enterotoxins of enterotoxigenic E. coli (ETEC), and have revolutionized the study of these pathogens [14]. Probes are now available for all diarrheagenic categories, although their usefulness varies. The aggregative adherence (AA) probe for E A E C , for example correlates strongly with HEp-2 adherence in some parts of the world, but not in others [15]. Diarrheagenic E. coli can be broadly categorized into three main classes: 1. Enterotoxigenic E. coli (ETEC); 2. Enteroadherent E. coli, including attaching-and-effacing forms-Enteropathogenic and Shiga toxin-producing E. coli (EPEC and STEC), as well as non-effacing subtypes (EAEC and diffusely adherent E. co/7-DAEC); 3. Enteroinvasive E. coli (EIEC), which invades the intestinal mucosa in a manner similar to Shigella. However, overlap among the categories exists. For example, both STEC and E A E C express toxins that contribute to disease, and some isolates of E A E C and ETEC exhibit invasiness. Like most enteric pathogens, E. coli follows a pattern of infection: colonization of a mucosal site, evasion of host defenses, multiplication, and host damage. An outline of mechanisms involved in bacterial infection of human epithelium are shown in Figure 1. A characteristic feature of diarrheagenic E. coli is their ability to colonize the host intestinal mucosal surface despite local defenses such as gastric acid, peristalsis, and competition from other microbial gut flora. 6 EXPOSURE to Pathogens Further Exposure at local sites INVASION 4 / COLONIEATION aid GROWTH Production of Virulence &ctors TOXICITY: toxin effects are local or systemic INVASIVENESS farther growth at original site and/or distant sites TISSUE DAMAGE: DISEASE Figure 1: An outline of the mechanisms of bacterial infection in human epithelium (Walker RI et al 1990) 7 This is partly attributable to surface fimbriae for mucosal adherence, which are expressed by almost all E. coli strains, including non-pathogenic species. Diarrheagenic E. coli possess specific fimbrial antigens that increase their ability to colonize the intestine and adhere to the small bowel mucosa, a site which is not normally colonized [16]. 1.5 Pathogenesis of Enteroaggregative Escherichia coli (EAEC) diarrhea E A E C is an emerging enteric pathogen that is distinguished from other E. coli by its ability to adhere to HEp-2 cells in an aggregative manner [17]. This aggregative adherence is characterized by prominent agglutination of the bacteria to each other, and typically, layering of bacteria in a stacked-brick configuration [8]. This pattern of adherence has been observed in human tissue explants and T84 intestinal epithelial cells in culture (Figure 2). In developed countries, E A E C diarrhea is most frequently identified in children attending day-care, in travelers and in immunocompromised individuals. E A E C is primarily recognized as a cause of endemic and persistent diarrhea in children residing in developing areas. It is also a major cause of traveler's diarrhea [18]. E A E C has also been implicated in several diarrheal outbreaks. The first of these occurred in Serbia [19], in which 19 infants in a nursery developed watery diarrhea. The largest outbreak occurred in 1993 in Japan, affecting nearly 2700 children who became ill after consuming contaminated school lunches [20]. Immunodeficient patients in developed countries have increased susceptibility to E A E C infection, and isolates in these patients are more likely to be resistant to conventional antibiotics [21]. In addition to causing diarrhea, E A E C infections in children are associated with intestinal inflammation and growth shortfalls, even in children without diarrhea [22]. 8 Figure 2: Stacked brick configuration of EAEC: Adherence of E A E C in the villous crypt (arrow-A, B & D) of human intestinal epithelium, and adherence to apical membrane of T84 intestinal epithelial cells in culture (arrowheads-C) (Nataro JP et al 1998). 9 Early childhood diarrhea correlates with long-term deficits in growth, physical fitness and cognitive function [23]. The long term effects of E A E C infection on growth and development of children in developing regions may thus be even more important than the short-term morbidity associated with diarrheal disease [22]. A prospective study of childhood diarrhea in an urban Brazilian slum found that E A E C was the leading cause of persistent diarrhea. Children from this study with E A E C and persistent diarrhea had significant elevations in fecal lactoferrin, interleukin-8 (IL-8) and IL-lp\ In addition, children with E A E C , but without diarrhea, had elevated fecal lactoferrin and IL-1B concentrations [22]. Histopathologic examinations of infected patients and studies in some animal models have established that E A E C increases mucus secretion from the intestinal mucosa, and forms a bacterium-mucus biofilm [16, 24, 25]. The role of excess mucus production in E A E C pathogenesis is not clear, but it may enable the bacterium to multiply and/or colonize the intestine. Alternatively, it is possible that mucus may confer certain advantages to E A E C , such as evasion from immunologic surveillance or protection from other intestinal flora. Unfortunately, animal models of E A E C diarrhea that replicate the hallmarks of infection have been not been developed to date. However, animal models of attaching and effacing (A/E) bacterial pathogens such as EPEC and EHEC are available. Citrobacter rodentium is the only known murine A / E pathogen, which serves as a useful model for studying EPEC and EHEC diarrhea in small animals [26]. A naturally occurring animal model of EHEC was developed in rabbits by Garcia et al to study the hemolytic uremic syndrome (HUS) [27]. 10 1.5.1 EAEC adherence and virulence factors A flexible, bundle-forming fimbrial structure known as aggregative adherence fimbrae I (AAF/I) mediates E A E C strain 17-2 adherence to HEp-2 and agglutination of human erythrocytes [28]. Nucleotide sequence analysis suggests that AAF/I are a member of the Dr family of adhesions [29]. A second fimbrial type, AAF/II, morphologically and genetically distinct from AAF/I, was subsequently identified [30]. The finding that isogenic AAF/II-negative mutants of strain 042 are no longer able to adhere to human intestinal explants in vitro [30] supports the relevance of AAF/II to clinical disease. Moreover, AAF/II was associated with E A E C diarrhea in children [31]. The role of other adherence factors and/or receptors in E A E C in forming the mucus biofilm is yet to be defined. Interestingly, IgA from human colostrum inhibits adhesion of E A E C to HEp-2 cells, suggesting that colostrum IgA antibodies reactive to E A E C antigens may play a role in protection of infants against diarrhea [32]. Other potentially pathogenic features of E A E C include invasion and toxin production. It has been suggested that some strains of E A E C may invade intestinal epithelial cells in vitro [33]. E A E C strain 236 has been reported to adhere to polarized Caco-2 and T84 intestinal epithelial cells and colonic mucosa in an in-vitro organ culture. Further, this strain invaded T84 cells and colonic mucosal explants and produced cytopathic effects [34]. The production of cytotoxins or other toxigenic mediators in E A E C infection has been studied in detail. EAST-1 is a 38 amino acid polypeptide homologous to STa of E T E C and is expressed by many E A E C strains. It is also expressed by 38% of non-pathogenic bacteria and its role in E A E C diarrhea remains undetermined [35]. Steiner et al described a 65kDa flagellin from E A E C (henceforth termed FliC-11 E A E C ) , which causes interleukin-8 (IL-8) release from Caco-2 intestinal epithelial cells [36]. Site directed mutagenesis undertaken to identify regions of F l i C - E A E C involved in IL-8 release has indicated that two non-adjacent regions in the conserved D l region of E A E C flagellin are necessary for induction of IL-8 secretion [37]. Amino acid sequence (386-407) in T L R 5 required for binding to Salmonella enteridis and E. coli flagellin has been identified [38]. Culture supernatants from certain E A E C outbreak strains contain a 104 kDa protein termed Pet (plasmid encoded toxin) that causes mucosal damage, mucus secretion and exfoliation of cells [39]. Pet is a member of the serine protease autotransporter toxin family. Upon internalization by host cells, it induces cytopathic effects of E A E C in some models [40]. Another factor that may play a role in aggregative adherence of E A E C has been reported recently. Dispersin is 10-kDa immunogenic protein secreted into the environment and possibly involved in adherence and colonization [41]. The authors of this study further propose that dispersin may facilitate the penetration of E A E C into the intestinal mucosal blanket to adhere and aggregate on the surface epithelium. The human colonic mucosa is covered with a continuous mucus layer, which is approximately 1 mm thick [42] and presents a formidable barrier against colonic pathogens. Large aggregates of enteric microbes would be retarded in their passage through the viscous gel. Therefore elaboration of flagellin and dispersin would theoretically facilitate the passage of E A E C and its subsequent binding to the host epithelial surface. A summary of E A E C virulence factors that have been identified to date is depicted in Table 1. 12 Table 1: E A E C virulence factors (Okeke IN et al 2001). Virulence marker type Virulence factor Prevalence in EAEC (%) Reference Plasmid-encoded toxins EAEC heat stable toxin 23 [43] Plasmid-encoded toxin+ 18-44 [44] Plasmid-encoded adhesins Aggregative adherence fimbriae I (AAF/I)+ 50-63 [28] AAF/II+ 12-46 [30] Outer membrane adhesins 18 kDa adhesin ND [45] 30 kDa haemaglutinin 14 [46] Pathogenecity islands Shigella she pathogenecity island (containing enterotoxin and mucinase genes) 1-57 [47] Yersinia high pathogenecity island 73 [48] (contains Yersinabactin siderophore gene) Extraintestinal E. coli hly pathogenecity island 3 [49] (contains hemolysin and P-pili genes) Locus of proteolysis activity island 20 [50] (shiga-toxin producing E.coli) Others Cytolethal distending toxin Rare [51] Pro-inflammatory flagellin ND [36] Shiga toxin Rare [52] Dispersin ND [41] ND=not determined, she=shigella-enterotoxin-encoding, E.coli hly-£.co//-alpha-hemolysin-encoding, + - unique to EAEC 13 1.6 The Innate immune system Epithelial cells at mucosal surfaces form the first line of defense against microbial pathogens. These cells are in a unique position of being in constant contact with bacteria and bacterial products, yet these factors that are normally pro-inflammatory for other cell types do not induce epithelial cells to initiate an inflammatory response. In the colon, the refractory nature of the epithelial cells to bacteria seems logical, as mounting an immune response to normal flora would be detrimental to the host. However, epithelial cells posses the ability to discriminate between pathogenic and non-pathogenic bacteria since infection of these cells by some pathogenic bacteria produces an inflammatory response [53, 54]. The innate immune recognition of bacterial products is an ancient system of host defense as there are striking similarities in the innate immune system of organisms as diverse as mammals, fruit flies and plants [55, 56]. The bacterial products that are recognized by these systems are conserved structural components of bacteria such as lipopolysaccharide (LPS) of Gram-negative organisms and peptidoglycan (PG) from cell walls of Gram-positive organisms [55, 56]. These microbial products are termed pathogen-associated molecular patterns (PAMPs). The innate immune system has developed a series of conserved receptors known as pattern-recognition receptors (PRRs), which recognize specific PAMP. This enables the innate immune system to distinguish self-molecules from pathogen-associated non-self structures and initiate the host defense response [57]. The recognition of PAMPs by PRRs results in the activation of signaling events that induce the expression of effector molecules such as cytokines, chemokines and co-14 stimulatory molecules, which subsequently control the activation of an antigen-specific adaptive immune response. Unlike the innate immune system, which recognizes PAMP non-specifically, the adaptive immune system is a specific response in the host. The adaptive immune system is mediated by B and T cells after exposure to the antigen, and is characterized by specificity, memory and self/non-self recognition. 1.6.1 Toll-like receptors An important advance in our understanding of early events in microbial recognition and the subsequent development of immune responses has been the identification of Toll-like receptors (TLRs) as the key PRRs of the innate immune system [57]. The gene that encodes for Toll protein was shown to be involved in embryonic development in Drosophila, and was identified subsequently as being an essential molecule for regulating the host immune response. TLRs are mammalian homologues of this protein that can detect PAMPs [58]. The first report of a mammalian TLR and its involvement in host defense was published in 1998 by Poltorak and colleagues, who discovered that mutations of the TLR4 gene selectively impede LPS signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin, yet highly susceptible to Gram-negative infection [59]. This finding was followed rapidly by the discovery that the human genome contains 10 distinct TLRs. Members of the TLR family share common characteristic extracellular and cytoplasmic domains. Their extracellular domains consist of several leucine-rich repeats (LRRs), whereas the cytoplasmic domain is similar to the interleukin-1 receptor (IL-1R) cytoplasmic domain, which is commonly referred to as the Toll/IL-IR homologous region (TIR domain) [60]. 15 1.6.2 TLRligands TLRs are expressed mainly in the cell types that form the first line of defense of the immune system, e.g. dendritic cells, macrophages, neutrophils, mucosal epithelial cells and dermal endothelial cells [61]. Different TLRs have been shown to be crucial for recognition of specific PAMPs that are common to a range of pathogens. The observation that a point mutation in the TIR domain of the mouse Tlr4 gene abolished the response to LPS provided the first evidence that this particular receptor might be involved in the innate immune response to Gram-negative bacteria [62]. By contrast, TLR2-deficient mice have a normal inflammatory response to LPS, but macrophages from these animals are less responsive to Gram-positive bacterial cell walls and peptidoglycan [63, 64]. These findings are evidence of TLR selectivity in PAMP recognition, although other TLRs can recognize the same components of both Gram-positive and Gram-negative bacteria. Flagellin, the principal element of bacterial flagella, is a highly virulent molecule that is recognized by TLR5 [65]. Some TLRs recognize bacterial products other than the components of the cell wall. For example, TLR9 is required for the inflammatory response that is initiated by hypomethylated bacterial DNA [66]. Recently, Takeuchi et al showed that TLR1 is involved in the recognition of mycobacterial lipoprotein and triacylated lipopeptides [67]. Some TLRs function by forming multimeric complexes (homodimers and heterodimers) to increase the spectrum of molecules that they recognize. For example, the cytoplasmic domain of TLR2 can form functional pairs with TLR6 and TLR1, leading to signal transduction and cytokine expression after ligand activation [60]. 16 Consistent with their role in protective immunity against microbial pathogens, each TLR appears to respond, either alone or in combination with specific microbial structures that represent PAMPs. The ligand(s) responsible for stimulating some TLRs have been identified [68] and include the following: For TLR2-lipoproteins, peptidoglycan, etc.; TLR3-poly I:C; TLR4-lipopolysaccharide; TLR5-flagellin; TLR7-nucleotide analogs such as imiquimod and TLR9-CpG motifs in bacterial DNA. LPS or endotoxin of Gram-negative bacteria, which induces endotoxin shock, is a prominent PAMP. LPS binds the soluble LPS binding protein (LBP) present in plasma [69], and the complex binds CD 14, which is a critical accessory molecule involved in LPS signaling [70]. CD 14 presents the LPS-LPB complex to TLR4 [71]. This triggers TLR4-mediated activation of several signaling molecules such as N F - K B , AP-1 [72], and p38 MAP kinase [73]. These signaling proteins are activated in target cells such as monocytes, macrophages and dendritic cells. In response to LPS these cells produce cytokines including IL-1, IL-6, IL-8, TNFa, and Thl associated cytokines such as IL-12 and IFNy. The involvement of extracellular-regulated kinase (ERKs), p38 MAP Kinase and Jun-N terminal kinase (JNK) in the LPS induced cytokine release has been extensively documented in several studies [74-76]. Complex formation with other molecules involved in pattern recognition such as CD 14 and MD2 has been implicated for TLR2 [77]. Furthermore, Toll-interacting (TOLLIP) and MyD88 (Myeloid differentiation factor 88) adapter like (MAL) proteins have been proposed to interact with TIR signaling domains and participate in signal propagation [78, 79]. It is not known if TLR5 signaling requires complex formation with accessory molecules. 17 1.7 Toll-like receptor (TLR) signaling pathways T L R s activate signaling pathways that are similar to those engaged by IL-1 as both share the TIR domain. This domain can interact with the adapter protein MyD88 , leading to activation of IL- lR-associated kinase ( IRAK) [60, 80]. I R A K , a serine-threonine kinase, activates tumor-necrosis factor receptor-associated factor 6 (TRAF6) [81]. Recruitment of T R A F 6 leads to activation of I-KB kinase ( IKK) which degrades I-KB , releasing N F - K B . Nuclear translocation of N F - K B leads to subsequent transcription of various pro-inflammatory genes for chemokines, proteins o f the complement system, enzymes such as cyclo-oxygenase-2 (COX-2) , inducible nitric oxide synthase ( iNOS), adhesion molecules and immune receptors [60]. These molecules are involved in engaging and controlling the innate immune response, which is essential for pathogen elimination and for mediating the transition to adaptive immune response. A schematic diagram of a proposed model of T L R 5 signaling pathway is shown in Figure 3. Another T L R signaling pathway was discovered by the observation that certain L P S induced responses do not require MyD88. This MyD88-independent T L R 4 signaling pathway was identified because N F - K B activation was delayed in MyD88-deficient cells, and was completely suppressed in T L R 4 deficient cells [82, 83]. These studies led to the identification of a new molecule known as the TIR-domain-containing adaptor protein (T IRAP, also known as M A L for MyD88-adaptor-like protein), which interacts specifically with T L R 4 , but not with other T L R s , and is probably responsible for MyD88-independent signaling. Horng et al have recently generated mice deficient in the Tirap gene [84]. TIRAP-deficient mice respond normally to the T L R 5 , T L R 7 and T L R 9 ligands, as wel l as to IL-1 and IL-18, but have defects in cytokine production and activa-18 T L R S Adaptive Immune response, Apoptosis & Antimicrobial response Figure 3: Proposed Toll-like receptor 5 signaling pathway (Axbibe et al 2000 and Hayashi et al 2001). P-phosphorylation and activation 19 -tion of N F - K B and MAP kinases in response to LPS. In addition, TIRAP-deficient mice are also impaired in their responses to ligands for TLR2, TLR1 and TLR6. Thus, TIRAP is differentially involved in signalling by members of the TLR family and may account for specificity in the downstream signaling of individual TLRs. The differences in signaling patterns could open up possibilities of selectively interfering with specific signaling components unique to particular TLR family members. A decline of TLR expression and function in macrophages from aged mice was observed in comparison to younger animals [85]. Both splenic and activated peritoneal macrophages from aged mice expressed significantly lower levels of all TLRs. Furthermore, macrophages from aged mice secreted lower levels of IL-6 and TNFa when stimulated with known ligands for TLR1 and 2, TLR3, TLR4, TLR5, and TLR9 when compared with those from young mice. These results support the concept that increased susceptibility to infections and poor adaptive immune responses in aging may be due to the decline in TLR expression and function [85]. TLR activation does not always lead to production of an immune response mediated by pro-inflammatory mediators. A characteristic of the three human-pathogenic Yersinia species (the plague agent Yersinia pestis and the enteric pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica) is the expression of the virulence antigen (LcrV). LcrV is a secreted protein that is involved in evasion of the host's innate immune response. A recombinant LcrV signals by activating toll-like receptor 2 (TLR2), leading to interleukin-10 (IL-10) mediated immunosuppression in mice [86]. The impact of this immunosuppressive effect for Yersinia pathogenesis is underlined by the observation that TLR2-deficient mice are less susceptible to oral Y. enterocolitica infection than isogenic 20 wild-type animals [86]. These data demonstrate that Yersiniae may exploit host innate pattern recognition molecules and defense mechanisms to evade the host immune response. 1.8 Mitogen-activated protein kinase (MAPK) signaling pathways Mammalian M A P K pathways can be activated by different stimuli acting through receptor tyrosine kinases (e.g. insulin and epidermal growth factor), vasoactive peptides (angiotensin II and endothelin), transforming growth factor (TGF)-p, and cytokines of the TNF family, and environmental stresses such as osmotic shock, ionizing radiation and ischemic injury. M A P K pathways, in turn, coordinate activation of gene transcription, protein synthesis, cell cycle machinery, differentiation and cell death [87, 88]. All M A P K pathways include a central three-tiered core signaling molecule in which M A P K are activated by tyrosine and threonine phosphorylation within a conserved Thr-X-Tyr motif in the activation loop of the kinase domain. A family of dual specificity kinases catalyzes their activation and phosphorylation. These dual specificity kinases are referred to as MAPK/extracellular-signal-regulated kinase (ERK)-kinases (MEKs or MKKs). MEKs in turn, are regulated by serine-threonine phosphorylation within a conserved motif of a kinase domain of several protein kinases. These are collectively known as MAP kinase kinase kinases (or MAP3Ks). M A P K core signaling molecules are themselves regulated by a wide variety of upstream activators and inhibitors [87, 88]. Stress activated protein kinase (SAPK)/JNK, p38 and ERK are the three important members of the M A P K signaling pathway activated by environmental stress and inflammatory cytokines. p38 MAP kinase was originally described as a 38-kDa polypeptide that underwent Tyr phosphorylation in response to endotoxin treatment and 21 osmotic shock [89]. The a-isoform of p38 M A P K was isolated by antiphosphotyrosine immunoaffinity chromatography. Subsequent cDNA cloning revealed that p38 was the mammalian homolog of HOG1, the osmosensing M A P K of S. cerevisiae. Most notably, the p38s, like HOGlp, contained the phosphoacceptor sequence Thr-Gly-Tyr [89]. Independently, two groups identified p38a as a kinase activated by stress and IL-1 that could phosphorylate and activate MAPK-activated protein kinase-activated protein kinase 1 (MAKAP kinase 1), a novel Ser/Thr kinase implicated in the phosphorylation and activation of the small 27-kDa heat shock protein HSP27 [90]. Of potential clinical importance, p38a was also purified and cloned as a polypeptide receptor for a class of experimental pyridinyl-imidazole anti-inflammatory drugs, the cytokine-suppressive anti-inflammatory drugs (CSAIDS), the most intensively characterized of which is the compound SB203580 [91]. CSAIDS were originally identified in a screen for compounds that could inhibit the transcriptional induction of TNF and IL-1 during endotoxic shock [91]. The basis for the efficacy of these compounds as anti-inflammatory agents was their ability to bind to and directly inhibit a subset of p38s, thereby blocking p38 mediated activation of AP-1, a trans-acting factor required for TNFoc and IL-1 induction. With the identification of additional p38 isoforms, four p38 genes are now known: p38ct, p38p\ p38y and p388 [92]. In vitro assays have demonstrated that only p38a and p38(3 are inhibited by CSAIDS; p38 y and p388 are completely unaffected by these drugs in-vitro or in transfected cells [93]. 1.8.1 Transcription factors regulated by p38 MAP kinase Max is a 12-kDa polypeptide that interacts with a transcription factor c-Myc enabling it to transactivate a subset of target genes. c-Myc is a key regulator of cell 22 proliferation, differentiation and apoptosis. The biological functions of c-Myc are thought to depend in part on the polypeptide-binding partners with which c-Myc interacts and the regulation of these binding partners [94]. A COOH-terminally truncated R N A isoform of p38a, known as Max interacting protein 2 (Mxi2), was isolated in a yeast two-hybrid screen for Max interactions. Max is a substrate for Mxi2 or p38a [95]. The functional significance of p-38 catalyzed Max phosphorylation is unclear. The SAPK and p38 M A P kinase are the dominant stress activated kinases responsible for the recruitment of AP-1 [88, 96]. AP-1 is a heterodimer comprising of basic leucine zipper (bZIP) transcription factors (typically c-Jun and JunD) along with members offos (c-Fos) and ATF (ATF2) families. A l l bZIP transcription factors contain leucine zippers that enable homo- and heterodimerization, and AP-1 components are organized into Jun-Jun, Jun-Fos or Jun-ATF [96]. AP-1 heterodimers containing ATF transcription factors can bind to both TRE (12-0-tetradecanoylphorbol-13 acetate-TP A responsive element) and CRE (cAMP responsive element) [96]. AP-1 is an important fram'-activator for a number of stress responsive genes including IL-1, IL-2, TNFa [96, 97] and IL-8 [98, 99]. Recently, M A P kinases including p38 M A P kinase, have been reported to contribute to IL-8 secretion from TNFa stimulated intestinal epithelial cells, via a posttranscriptional mechanism involving the stabilization of IL-8 transcript [100]. Inflammatory stimuli induce p38 M A P kinase dependent phosphorylation and phosphoacetylation of histone H3 on promoter regions of chemokine and cytokine genes [101]. p38 activity was also required to enhance the accessibility of the cryptic N F - K B binding sites contained in H3 phosphorylated promoters which leads to increased N F - K B 23 recruitment. Furthermore, a role for p38 MAP kinase as a negative regulator of gene expression of some TLRs has been proposed in recent reports [73, 102]. In addition to cytokine release, some studies have investigated a role for p38 MAP kinase in the expression of TLRs. It was recently discovered that p38 MAP kinase plays an important role in the regulation of TLR2, TLR4 and TLR9 gene expression in mouse dendritic cells challenged with LPS [73]. Increased expression of TLR2 in epithelial cells greatly enhances the Non-typable Hemophilus influenza induced expression of several key cytokines including TNFcc, IL-ip and IL-8 [103]. IL-8 is a critical chemokine that is involved in the recruitment of neutrophils in acute and chronic inflammatory conditions [104]. Similarly, IFNy treatment of gingival and oral epithelial cell lines increased TLR2 and TLR4 expression leading to an increase in IL-8 and G M -CSF secretion upon exposure to LPS and bacterial polypeptides [105]. These findings indicate that IFNy and other cytokines may modulate the expression of TLRs by a mechanism that involves p38 MAP kinase activation. 1.9 Regulation of interleukin-8 (IL-8) transcriptional activation Stimulus dependent activation of IL-8 gene transcription has been demonstrated in nuclear run-on experiments [106, 107]. In a number of studies it was found that a sequence spanning -1 to -133 within the 5' flanking region of the IL-8 gene is essential and sufficient for the transcriptional regulation of the gene [108]. As demonstrated by mutational and deletional analysis, this promotional element contains a N F - K B element that is required for activation in all cell types studied. NF- KB is a dimeric transcription factor composed of a family of 5 subunits, namely NF- KB (p50 and its precursor pi 05), NF- KB2 (p52 and its precursor pi00), and c-REL, REL A (p65) and REL B [109]. By 24 using chromatin immunoprecipitation, binding of the p65 N F - K B to the endogenous IL-8 promoter and subsequent recruitment of RNA polymerase II are found rapidly, within 1 hour of IL-1 stimulation, underlining the importance of N F - K B in IL-8 transcription [109, 110]. The core IL-8 promoter also contains activating protein (AP-1) and CAAT/enhancer- binding protein (C/EBP) sites. The latter two sites are dispensible for transcriptional activation in some cells, but contribute to activation in others. Therefore, unlike the N F - K B binding site, the AP-1 and C/EBP sites are not always essential for induction but are required for maximal gene expression [104]. In contrast to N F - K B , AP-1 proteins are constitutively bound to their cognate DNA element. Transcriptional activity of AP-1 proteins is regulated by their abundance, phosphorylation of trans-activation domains, and by their binding to protein kinases [96, 111]. It is important to note that in contrast to N F - K B , whose p65 subunit binding to the IL-8 promoter has been analyzed at the atomic level [112], the composition of the endogenous AP-1 dimer as function of time and stimulus that modulates IL-8 transcription has not been determined. Little is known of the signaling pathways regulating C/EBPs, including the family member C/EBP-P (also known as NF-IL-6), which was found to bind to the IL-8 promoter [113, 114]. Inhibition of p38 MAP kinase by pyridylimidazole analogues SB 203580 and SB 202190 suppresses induction of IL-8 in peripheral blood mononuclear cells (PBMC), T84 and THP-1 cells [115]. Available data indicates that unlike N F - K B , p38 MAP kinase regulation of IL-8 is not essential in some systems such as human synovial fibroblasts and mast cells [116, 117]. In addition to regulating IL-8 mRNA, p38 MAP kinase also 25 influences IL-1 and IL-6 expression. This is supported by the observations made in p38 deficient mice. In these animals, the expression of IL-1 and IL-6 by TNFa was defective in comparison to wild type mice [118, 119]. p38 MAP kinase has also been shown to regulate a specific post-translational step in IL-8 gene expression [120, 121]. The low amount of IL-8 found in unstimulated cells is not only a result of repressed transcription but also due to very unstable mRNA. Adenine and Uracil bases in the IL-8 mRNA are rapidly degraded, mediated by the adenylate/uridylate-rich element (ARE), contained in its 3' untranslated region. It is interesting that this part of mRNA is also required for signal-mediated stabilization of mRNA transcripts. By measuring IL-8 mRNA stability with tetracycline regulatable reporter gene constructs [120, 121], p38 MAP kinase was found to stabilize IL-8 mRNA. Specifically, these data show that an active form of M K K , which selectively activates p38 MAPK, induced marked stabilization of IL-8 transcript. In brief, these findings indicate that the p38 MAP kinase pathway regulates IL-8 gene expression by stabilizing mRNAs by MK2 dependent mechanisms. In addition, the p38 MAP kinase pathway has been implicated in regulating the stability of nine ARE-containing transcripts, including TNFa, IL-1, IL-8, BCL-2 and MCSF-1 [122, 123]. ARE-mediated mRNA turnover is an important regulatory component of gene expression for innate immunity. These studies underline the importance of p38 MAP kinase in regulating stress-induced responses in epithelial cells. 1.10 Biological functions of IL-8 The ability of invasive pathogens to induce intestinal epithelial cells to secrete chemokines has provided important insights into mechanisms that initiate inflammation. 26 IL-8 was initially purified as a chemotactic factor for neutrophils [124]. However, subsequent studies demonstrated that IL-8 secretion in-vitro has multiple effects on neutrophils, including induction of shape change [125], release of lysosomal enzymes [126], induction of respiratory burst [127], and generation of superoxide and hydrogen peroxide [125]. IL-8 also induces generation of bioactive lipids [128] and increases the expression of adhesion molecules on neutrophils [129]. In addition to neutrophils, the chemotactic activity of IL-8 is exerted on basophils [130] and IL-3 or GM-CSF primed eosinophils [131], although the pathological significance of these effects remain elusive. The role of IL-8 in the pathogenesis of E A E C infection is unclear; however it is likely to involve chemotaxis. In one study, children with E A E C were found to have elevated levels of fecal lactoferrin, IL-8 and IL-1 p, which are markers of inflammation [22]. In another study, travelers to India who had EAEC-associated diarrhea had high levels of IL-8 and IL-ip, although travelers to Mexico who developed E A E C diarrhea were not found to have increased fecal cytokines [132]. Although most patients with E A E C diarrhea have elevated IL-8 levels in their stools, a subset of patients with E A E C diarrhea fail to develop an inflammatory response. Whereas markers of inflammation have been found in patients with E A E C diarrhea [22, 132], neutrophilic infiltration of large or small intestine has not been demonstrated in-vivo. Intestinal inflammation has been demonstrated in experimental Shigella flexneri infection in infected rabbit ligated intestinal loops [133]. S. flexneri enters the epithelial barrier essentially through the dome of lymphoid follicles at the early stage of infection and that subsequent invasion and destruction of the epithelium is primarily due to immigration of leukocytes, particularly PMN that destroy cohesion of the epithelial 27 barrier. FliC-EAEC-induced IL-8 secretion during E A E C diarrhea, is likely to result in intestinal inflammation by neutrophils chemotaxis. In the intestine, neutrophils are known to contribute to fluid secretion by releasing cAMP, which is metabolized to adenosine, activating enterocyte adenosine receptors [128]. 1.11 Mechanisms of inflammation in the human intestinal epithelium The immunological landscape of the mucosal surface of the gastrointestinal tract (GI) is extraordinary. In the colon, for example, the microbial burden is estimated to be 109 organisms per milliliter of the luminal content. Across the majority of the surface area of the GI tract, a single layer of epithelial cells separates the antigenic material from the underlying gut associated lymphoid tissue (GALT). In both anatomical and functional contexts, the structure of the intestinal epithelium poses a formidable barrier. In addition, epithelial cells produce copious amounts of mucus, and this along with glycocalyx limits both particulate antigen and bacterial contact with the apical surface of the cells. Furthermore, epithelial cells secrete anti-microbial peptides and proteins that limit bacterial colonization [134]. Over the past decade it has become increasingly clear that intestinal epithelial cells (IEC) not only provide a critical barrier function but also are also active in mucosal immune responses. For example, IECs secrete and respond to wide array of cytokines, chemokines and other immunologically active molecules [135]. In addition, IECs maintain intimate contact with various populations of interdigitating, bone marrow-derived lymphoid and myeloid cells including T cells and polymorphonuclear cells, which are recruited to the site of inflammation. In this context, IECs are known to express human leukocyte antigen (HLA) class 1, H L A class II, and a number of H L A 28 class I-like antigen presenting molecules like M I C A and MICB [136]. Furthermore, IECs express a variety of immunologically relevant molecules on their surface, including several TLRs that play a critical role in immune responses. Although differences in signaling pathways between TLRs are emerging, TLR4 activation involves PI-3K/Akt and I-KB in non-transformed rat intestinal epithelial cells (IEC-6) due to invasive Gram-negative Bacteroides vulgatus [137]. Expression of TLR4 and MD-2 was also investigated in normal colonic epithelial cells and intestinal epithelial cell lines. The effect of the cytokines IFNy, IFNa, and TNFa on TLR4 and MD-2 expression was examined by RT-PCR and Western blot [138]. IFNy was observed to regulate MD-2 expression in both IEC lines, whereas IFNy and TNFa regulate TLR4 mRNA expression in IEC lines. Pre-incubation with IFNy and/or TNFa sensitizes IEC to LPS-dependent IL-8 secretion. T cell-derived cytokines lead to an increase in expression of TLR4 and MD-2, as well as LPS-dependent pro-inflammatory cytokine secretion in IEC. IFNy regulates expression of the critical TLR4 co-receptor MD-2 through the Janus tyrosine kinase (JAK)-STAT pathway. These findings suggest a role for Thl cytokines, which may initiate or perpetuate intestinal inflammation by altering toll-like receptor expression and bacterial reactivity [138]. As noted above, Hayashi et al reported in 2001 that flagellin from Gram-negative bacteria activated TLR5 leading to N F - K B mediated TNFa production in CHO cells [65]. However, it is not known if TLR5 activation is involved in inflammatory responses in the large intestine during E A E C diarrhea. Epithelial cell lines such as mouse primary renal tubular cells have been shown to express TLRs 1,2,3,4 and 6 but not TLR5 or 9. These cells release chemokines such as monocyte chemoattractant protein (MCP-1) and 29 RANTES in response to LPS. Inhibitors of N F - K B abolished the release of these chemokines, whereas inhibitors of ERK did not affect chemokine release [139]. 1.12 Toll-like receptors (TLRs) and the intestinal epithelium It is not known if the mechanisms outlined above are activated due to Gram-negative flagellin in intestinal epithelial cells. Hobbie et al investigated the interaction of the Salmonella type III secretion system with the intestinal epithelium [115]. This secretion system causes activation of N F - K B and AP-1, resulting in the production of pro-inflammatory cytokines such as IL-8. The study also showed that S. typhimurium infection of cultured intestinal epithelial cells results in the activation of ERK, J N K , and p38. Induction of these signaling pathways and the synthesis of IL-8 were strictly dependent on the function of the invasion-associated type III protein secretion system encoded by S. typhimurium. Pretreatment of cells with SB 203580 prevented S. typhimurium-induced IL-8 production. These results indicate that the inflammatory response induced by S. typhimurium may be due to the specific stimulation of MAP kinase signaling pathways leading to nuclear responses [115]. Recently, activation of TLR5 signaling pathways involving p38 MAP kinase activation by S. enteridis flagellin have been described in Caco-2 cells [164], which suggest that invasion of epithelial cells is not necessary for activation of p38 MAP kinase. The release of IL-8 in response to LPS in intestinal epithelial cells was found to correlate with TLR 4 expression in undifferentiated HT-29 cells but not to expression of TLR2 or CD 14 [140]. Although CD 14 was expressed constitutively by Caco-2 cells and differentiated HT-29 cells, these cells were not responsive to LPS [140]. 30 The inflammatory effect of Salmonella flagellin was studied by Eaves-Pyles in intestinal epithelial cells. In Caco-2 and DLD-1 cells, Salmonella flagellin induced I-KB degradation, N F - K B nuclear migration and iNOS expression. In the same study, when flagellin was injected systemically in mice at low doses, it produced systemic inflammation characterized by release of pro-inflammatory cytokines, chemokines and iNOS. At higher doses, flagellin induced shock, characterized by hypotension, reduced vascular contractility in mice, and death. The effects of flagellin do not diminish in C3H/HeJ LPS-resistant mice (deficient in TLR4), indicating that Salmonella flagellin-induced signaling is independent of TLR4 and LPS [141]. Another subject of active investigation has been the localization of TLRs in the intestinal epithelium. Central to this controversy is the hypothesis that expression of TLRs on the epithelial surface of the gut, which contains a sea of microbes and microbial products, constitutes a significant risk in the development of exaggerated or chronic inflammatory responses within the intestinal mucosa. In this setting, the mechanism by which IEC's express TLRs could regulate their ability to respond, or not to respond to inflammatory stimuli. One possible explanation would be expression of TLRs at a site away from the apical surface of the gut epithelium. For example, Gewirtz et al reported that bacterial flagellin activates basolaterally expressed TLR5 in T84 cells to induce pro-inflammatory gene expression [142]. Similarly, TLR4 was expressed in the Golgi apparatus and colocalized with internalized lipopolysaccharide in murine intestinal epithelial cells m-IC (cl2) [143]. The growing literature on the functional expression of TLR4 on polarized IECs raises the possibility that TLR5 may be expressed in a similar 31 manner within IECs, because flagellin is continuously present as a component of bacteria in the gut flora. 1.13 The relevance of TLR signaling pathways The discovery of TLRs has provided us with an insight into the mechanisms by which the innate immune system senses and responds to pathogens and subsequently influences the outcome of an adaptive immune response in the host. These new insights not only provide a basis for development of new therapies but also offer the potential to develop disease-modifying therapies that would regulate the induction of T helper cells from a phenotype that results in allergic responses (Th2) to one that eradicates disease (Thl). Moreover, reagents that enhance TLR signaling pathways can be powerful adjuvants for fighting pathogens or cancer [144]. Recent data lend support to the extended role of TLRs by showing that, in addition to the pathogen derived signals, endogenous ligands such as surfactant protein A [145], Heat shock protein 60 - HSP60 [146] and Hyaluronic acid-HA fragments [147] can activate TLRs. Interestingly, not only bacterial but also mammalian HSPs interact with TLRs demonstrating that the exclusive association of TLRs with microbial ligands is an obsolete concept. Human HSP60 and Gp96 (another heat shock protein) are the first examples of non-pathogen derived ligands of TLRs. More importantly, Gp96 provides the first example of the innate and adaptive immune system being stimulated simultaneously by the same molecule, which is released under physiological conditions from necrotic cells [148]. These data suggest that TLRs play an important role in the maintenance of the inflammatory process that lead to acute and chronic inflammation. Compounds such as 32 oligonucleotides, fatty acids and imidazoquinolones acting at various TLR, have an important implication from the point of view of the pharmaceutical industry. Indeed, the demonstration that a low-molecular mass compound, resiquimod [149], can activate TLR7 and TLR8 and has anti-viral and anti-allergic properties supports the concept that targeting TLRs could offer a promising new therapeutic strategy for the treatment of human inflammatory diseases that are caused by dysregulation of the immune system. In summary, TLR signaling in IEC is a critical component of the innate immune response and may subsequently modulate the development of the adaptive immune system in a growing child. A deficient TLR signaling pathway may produce abnormal responses to pathogens, and lead to chronic inflammation and/or growth impairment in children. 1.14 Hypothesis and objectives The mechanisms of signaling induced by F l iC-EAEC during IL-8 secretion from intestinal epithelial cells have not been characterized previously. Further, the role of TLR5 in this reponse is unclear. The objective in this thesis is to study the signaling mechanisms of IL-8 secretion in intestinal epithelial cells and identify the principal mediators of chemokine secretion. The activation of PI-3K by T N F a has been shown in colonic epithelial cells [167]. Further, activation of TLR leads to N F - K B and M A P kinase-mediated chemokine gene expression [65, 73]. Therefore, studies in this project will focus on PI-3K and p38 M A P kinases as possible regulators of FliC-EAEC-induced IL-8 secretion in epithelial cells. The hypothesis of this project is: 33 Enteroaggregative Escherichia coli flagellin (FliC-EAEC)-induced IL-8 release is mediated by TLR5 and dependent on PI-3K and p38 M A P kinase activation in intestinal epithelial cells. The specific aims of this project are as follows: 1. To determine the role of TLR5 in FliC-EAEC-induced IL-8 secretion from human epithelial cells. 2. To verify whether FliC-EAEC-induced IL-8 secretion is dependent on p38 M A P kinase activation in epithelial cells. 3. To verify wheather PI-3K signaling pathways regulate FliC-EAEC-induced IL-8 secretion. The experiments in this thesis will attempt to answer the following questions: What are the signaling mechanisms that regulate the F l iC-EAEC induced secretion of IL-8 from intestinal epithelial cells? Does Fl iC-EAEC induced IL-8 secretion requires expression of TLR5 and activation of PI-3K and/or p38 M A P kinase in epithelial cells? To answer the above questions, studies will be undertaken in transformed human cell lines as a model of host-pathogen interaction or infection in vitro. Initially, the effects of Fl iC-EAEC on PI-3K and p38 M A P kinase signaling pathways will be determined by in-vitro kinase assay and Western immunoblotting in Caco-2 intestinal epithelial cells. Subsequently, pharmacological inhibitors of PI-3K and p38 M A P kinase will be used to inhibit IL-8 secretion by Fl iC-EAEC. Monocytic cells (THP-1) will be studied to determine i f these cells secrete IL-8 in response to F l iC-EAEC. The rationale for including THP-1 cells in our study is that previous studies have shown IRAK activation in these cells upon exposure to Gram-34 negative flagellin [150]. Our aim will be to reproduce our findings in THP-1 cells incubated with Fl iC-EAEC. The role of TLR5 in Fl iC-EAEC signaling will be investigated in epithelial cells transiently transfected with TLR5. This approach will hopefully lead to identification of a dominant signaling protein that is critical for FliC-E A E C induced IL-8 release. 1.15 Caco-2 intestinal epithelial cells: a model for studying intestinal inflammation Caco-2 cells are a human adenocarcinoma cell line that forms polarized monolayers and well defined brush borders in culture. These cells express several markers characteristic of small intestine villous morphology and develop high levels of trans-epithelial electrical resistance identical to the human intestinal epithelium [151, 152]. In 1987, the first report of a mechanism of bacterial infection in Caco-2 cells was published. In this study, the invasion and replication of Listeria monocytogenes in intestinal epithelial cells were examined by infecting Caco-2 cells [153]. Since then, the interaction of Gram-negative bacteria with Caco-2 cells has been investigated by other groups [151, 154], making this cell line a useful model of intestinal infection in-vitro. The expression of a receptor for E. coli heat stable enterotoxin in Caco-2 cells was published in 1993 [155, 156]. Enterotoxin-receptor interaction caused activation of guanylyl cyclase in Caco-2 cells [155], indicating that bacterial toxins stimulated intracellular enzymes in these cells. Various other proteins and mature enterocyte-linked functions of normal intestine have been observed in Caco-2 cells [157]. These include receptors for L D L [158], IGF-I and II/M6P [159], Vasoactive intestinal peptide (VIP) [160] and enzymes such as sucrase-isomaltase [161]. Cario et al have described TLR2 and TLR4 protein expression in Caco-2 cells [162]. TLR5 mRNA has been demonstrated 35 in epithelial cell lines derived from the intestine, lung and kidney [163]. Others have shown that Salmonella flagellin (TLR5 ligand) causes IL-8 secretion from Caco-2 cells, which is an indirect evidence of TLR5 expression in these cells [164]. The secretion profile of cytokines from Caco-2 cells is well defined. Schuerer-Maly et al examined the characteristics of IL-8 release from four intestinal epithelial cell lines [165]. These investigators discovered that colonic adenocarcinoma cell lines T84, HT29 and S W620 cells all secreted substantial amounts of IL-8 when stimulated with IL-ip, TNFa and IFNy whereas Caco-2 cells only secreted IL-8 in response to IL-1 p. Stimulation of Caco-2 and other intestinal epithelial cells with IL-1 P and TNFa leads to increased expression of IL-8 and TGFp mRNA [166]. An advantage of Caco-2 cells compared to other IECs is their unresponsiveness to LPS. Studies have shown that Caco-2 cells do not secrete IL-8 when stimulated with LPS [165]. While LPS causes a dose-dependent secretion of IL-8 in undifferentiated HT-29/p cells, Caco-2 cells were found to be unresponsive, even when constitutively expressing mRNA transcripts for CD 14 [140]. In this study, Fl iC-EAEC signaling was investigated in Caco-2 cells with the aim of elucidating the roles of PI-3K and p38 MAP kinase. These kinases are known to be involved in wide range of cellular processes associated with inflammation and other cellular functions [167, 168, 115]. Further, MAP kinases such as p38 are activated by Salmonella and EHEC infection of intestinal epithelial cells [115, 169]. 1.16 THP-1 promonocytic cell line: a model for studying host-pathogen interaction The human leukemic cell line-THP-1 was derived from a boy with acute monocytic leukemia. This cell line has Fc and C3b receptors, but no surface or 36 cytoplasmic immunoglobulins. The monocytic nature of the cell line was characterized by: (1) the presence of alpha-naphthyl butyrate esterase activity, which could be inhibited by NaF; (2) lysozyme production; (3) the phagocytosis of latex particles and sensitized sheep erythrocytes; and (4) the ability to restore T-lymphocyte response to Con A [170]. The THP-1 cell line is a useful monocytic model system in which to study pro-inflammatory cytokine production in response to stimulation with bacterial proteins [72, 171]. The intestinal epithelium that lines the gut is a mucosal barrier that detects and responds to the antigenic molecules in liaison with underlying immune cells. Cross talk with the underlying gut associated lymphoid tissue (GALT) activates the innate and acquired immune system of the host. It is now clear that the microenvironment in this mucosal barrier has a marked influence on the immune response that ultimately ensues. The G A L T must therefore, constantly distinguish harmless antigens that are present in food or in commensal bacteria from pathogenic assault by microbes [172]. Since Caco-2 intestinal epithelial cells respond to Fl iC-EAEC by producing IL-8, it is conceivable that this chemokine forms part of an inflammatory response that is relayed to G A L T , which constitutes the secondary lymphoid tissue comprising of macrophages, dendritic cells, B and T-lymphocytes. We decided to study THP-1 cells to determine if human monocytic cells release IL-8 after exposure to Fl iC-EAEC. Since S. enteridis flagellin has been shown to activate IRAK in these cells [150], studies in THP-1 cells would support our hypothesis that Fl iC-EAEC activates TLR5 signaling pathway. It is known that monocytic cells respond to Gram-negative flagellin by producing inflammatory cytokines. For example, S. typhimurium and P. aeruginosa flagellins are 37 potent inducers of TNFa from human monocytes and THP-1 cells [173]. While, IRAK has been implicated in flagellin signaling in THP-1 cells, it is not known i f this leads to N F - K B and p38 M A P kinase activity. To date, the signaling mechanisms distal to TLR/IRAK remain unknown in THP-1 cells. Northern analysis has demonstrated that monocytic cells, such as THP-1 express mRNA for TLR5 [174]. Although flagellin signaling is not well defined in THP-1 cells, endotoxin-induced signaling via TLR4 has been studied in greater detail [175, 176]. Regulation of TLR mRNA expression in response to a variety of stimuli including LPS and bacterial lipoproteins has been observed in THP-1 cells. Furthermore, exposure of P B M C to E. coli caused distinct changes in expression of TLRs, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation [177]. Flagellin signaling is believed to influence the profile of cytokine secretion from monocytic cells or macrophages, eventually altering the balance of Thl vs. Th2 cytokines in the host upon subsequent exposure to microbial peptides [144]. The immunostimulatory effects of flagellin during shock and septicemia are thought to involve TLR5 and N F - K B [178], although data for a direct correlation between the two are currently lacking in monocytic cell lines. 1.17 Human epithelial HEp-2 cell line: a model for studying host immune responses The specific aims of studies in HEp-2 cells were: 1. To establish that F l iC-EAEC activates p38 M A P kinase in TLR-transfected HEp-2 cells. 38 2. To verify that FliC-EAEC-induced IL-8 secretion from TLR5-transfected HEp-2 cells is regulated by p38 MAP kinase. 3. To determine if Fl iC-EAEC induces IRAK activation in TLR5-transfected HEp-2 cells. HEp-2 cells are a transformed cell line derived from an epidermoid carcinoma of the larynx [179]. The HEp-2 cell adherence assay was first used by Cravioto et al 1979 to study adherence among EPEC strains from large outbreaks [11]. They found that 80% of EPEC exhibited adherence to HEp-2 tissue culture cells, whereas most strains of E.coli from subjects without diarrhea were not adherent. Adherence of EPEC to HEp-2 cells is a well established model used for studying mechanisms of pathogenesis [180-182]. Using this model, Baldini et al had earlier shown that adherence was mediated by a 50-70 kDa plasmid in most strains of EPEC, whereas HB101, a nonadherent E. coli, acquires HEp-2 adhesiveness after gaining this plasmid. Plasmid presence was also shown to correlate with in vivo adhesion to intestine, using the colostrum-deprived gnotobiotic piglet model [183]. However, these studies also showed that many E. coli strains other than EPEC serogroups also adhered to HEp-2 and that the adherence phenotype was clearly distinguishable from that of EPEC. The adherence pattern of EPEC was described as "localized adherence". In contrast, non-EPEC pathogens did not adhere in characteristic micro-colonies and instead displayed a phenotype initially described as "diffuse adherence" [180]. The adherence properties of E. coli isolates were further analyzed and were subsequently distinguished as aggregative and diffuse adherence phenotypes [17]. Aggregative adherence (AA) is defined by prominent auto-agglutination of the bacterial cells to each other, either on the 39 surface of HEp-2 cells, or on glass cover-slip free from HEp-2 cells. It is not known if adherence of E A E C causes activation of pro-inflammatory cytokine signaling. Gram-negative bacteria are known to cause release of cytokines from HEp-2 cells by mechanisms involving N F - K B activation. S. typhimurium induces membrane ruffling and its own internalization by a signaling pathway that is independent of Rac and Epidermal growth factor-receptor, in HEp-2 cells [184]. Co-culture of strains of Helicobacter pylori with HEp-2 cells led to secretion of IL-8 [185], suggesting that HEp-2 cells mount inflammatory responses to bacterial factors. To date, no TLR's have been demonstrated in HEp-2 cells and there is no evidence of M A P kinase activation by microbial products in this cell line. However, it is reasonable to assume that some elements of TLR signaling machinery is functional in laryngeal epithelial HEp-2 cells, since a related cell line, bronchial epithelial- 16HBE14 cells, release IL-8 in response to neutrophil elastase, by activating I R A K signaling pathway [186]. Unlike LPS, F l iC-EAEC does not induce IL-8 secretion from HEp-2 cells in their native (untransfected) state. However, transient expression of TLR5 in these cells led to an increase in IL-8 secretion upon exposure to F l iC-EAEC [37]. Therefore, we decided to investigate the role of TLR5 signaling pathways in secretion of IL-8 from transfected HEp-2 cells exposed to F l iC-EAEC. 40 Chapter II: Materials and Methods 2.1 Caco-2 cell culture for studies of PI-3K and p38 MAP kinase activation by FliC-EAEC Caco-2 cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD) and grown in Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L D-glucose, lx non-essential amino acids, 1 x Sodium Pyruvate, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 ug/ml) (Sigma Aldrich Canada), and 10% fetal bovine serum (FBS). (Fisher Scientific, Canada). Caco-2 cells were seeded at a density of 2 XI0 6 in 6 well polystyrene plates (VWR International) and cultured at 37 °C for various time-points in a humidified atmosphere of 5% CO2 in a DH-Autoflow incubator (Nuaire, Canada). 5-7 days after cells became confluent, they were treated with FliC-EAEC. 2.2 THP-1 cell culture for studies of IL-8 secretion, PI-3K and p38 MAP kinase activation by FliC-EAEC The monocytic cell line THP-1 was kindly provided by Dr. Z. Hmama (Division of Infectious Diseases, University of British Columbia) and cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 ug/ml). Cells were seeded at a density of 2 X 106 per well in 6 well plates, subsequently treated with Fl iC-EAEC and incubated at 37°C for various time-points in a humidified atmosphere of 5% CO2. 2.3 Transient expression ofTLR5 in HEp-2 cells pEF6/V5-His containing the full-length human TLR5 gene was a gift from A. Aderem (University of Washington, Seattle, WA). pEGFP-Nl vector (Clontech, Palo 41 Alto, CA) expressing the green flourescent protein (GFP) was used as a transfection control. HEp-2 cells were cultured in Ham's F12 medium with penicillin (100 U/ml), and streptomycin (100 ng/ml) and 5 % FBS. Prior to transfection, HEp-2 cells were released with 0.25% trypsin/EDTA (Sigma-Aldrich Canada) and seeded at 105/ well in 12-well polystyrene dishes (VWR International). After 24-48 h, the medium was replaced with F12 without serum. Cells were then transfected with DNA using 0.5 u.g of each plasmid and 6.6 pi of ExGen-500 polyethylenimine reagent (MBI Fermentas) per well. Plates were centrifuged for 5 min at 1100 rpm, incubated for 4-6 hrs, and then fed with serum containing growth medium. Expression of GFP was confirmed at 48 h by fluorescence microscopy. Cells were fed with 500 ul of fresh growth medium, and Fl iC-EAEC samples added. Supernatants were removed after 3 h of incubation and IL-8 concentration assessed by ELISA (BD Biosciences). For signaling studies, cells were treated with Fl iC-EAEC for various time points, lysed, and whole cell lysates subjected to Western analysis. 2.4 IL-8 secretion from U937 leukemic cell line incubated with FliC-EAEC The U937 promonocytic cell line derived from human histiocytic lymphoma is studied as a model of infection and inflammation [187]. U937 cells transfected with cDNA encoding the entire coding region of either wild-type bovine PI-3K subunit p85oc (Wp85a) or mutant bovine p85a (Ap85a) has been described [188]. The mutant has a deletion of 35 amino acids from residues 479-513 of bovine p85ct and the insertion of two amino acids (Ser-Arg) in the deleted position. Mutant p85a competes with native p85 for binding to essential signaling proteins, therefore acting as a dominant negative mutant [189]. This cell line was utilized for studying the involvement of PI-3K in FliC-42 E A E C induced IL-8 secretion. U937 cells (gift by Dr. Neil Reiner) were cultured in RPMI 1640 supplemented with 10% FBS, 2mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 ug/ml). Cells were seeded at a density of 106 per well in a 6 well plates, subsequently treated with FliC- E A E C and incubated at 37°C for various time-points in a humidified atmosphere of 5% CO2. 2.5 Expression and purification of EAEC flagellin The full-length gene encoding the E A E C flagellin fliC-EAEC [36] with an N -terminal 6XHis tag was maintained in ToplOF' (Invitrogen Life technologies, Canada). The pfliC-EAEC vector has gene for ampicillin resistance gene allowing selection of transformed bacteria containing this plasmid. CaC^-competent BL21 (DE3) pLysS cells with a gene for chloramphenicol resistance were used for tranformation and expression of FliC-Histidine tagged protein [36]. For cloning, a 250 ul vial of BL21 pLysS was thawed on ice and lui of pTSS8 vector was added and mixed gently. Cells were incubated on ice for 30 minutes and then heat shocked for 1 min. at 42 °C in a water bath. The cells were then placed immediately on ice and 1ml of sterile LB (Laurea Bertani) broth was added. This pre-culture was transferred to sterile polypropylene round bottom tubes and incubated at 37 °C for 1 hour. Subsequently, this was poured into sterile 50 ml conical tubes containing 10 ml LB, ampicillin 100 ug/ml (Sigma Aldrich Canada), chloramphenicol 34 |ig/ml (Fisher Scientific) and incubated overnight at 37 °C incubator. Log-phase cultures were induced with 0.5 to 1.0 mM isopropyl-fl-D-thiogalactopyranoside (IPTG) (Sigma Aldrich Canada) for 4 h at 37 °C in 2 L conical flasks. Cultures were then transferred to 250 ml centrifuge bottles and pelleted by 43 centrifugation at 7000 x g at 4 °C in a Sorvall * RC-5B ultracentrifuge (Mandel). Bacteria were lysed by two freeze-thaw cycles followed by sonication (three 10 second pulses) using a Sonic Dismembrator 60 Sonicator (Fisher Scientific), and lysates were cleared by centrifugation (15,000 x g for 20 min at 4 °C). Cleared lysates were then tested for Caco-2 cell IL-8 release. Recombinant His-tagged flagellin was purified under native conditions by Cobalt affinity chromatography (Talon resin; Clontech Laboratories, Inc.). Immobilized Metal Affinity Chromatography (IMAC) was first introduced in 1975 as technique for separating proteins [190]. The principle is based on reversible interaction between various amino acid side chains and immobilized metal ions. Depending on the immobilized metal ion, different side chains can be involved in the adsorption process. Most notably, histidine, cysteine and tryptophan side chains have been implicated in protein binding to immobilized transition metal ions and zinc [190, 191]. Protein expression was verified by electrophoresis on 9% SDS-PAGE followed by staining with EZ-Blue™ gel staining reagent (Sigma Aldrich Canada) to identify a single Fl iC-EAEC band corresponding to 65kDa, which was taken as evidence of purified EAEC-flagellin protein. E A E C flagellin was diluted in PBS (Sigma Aldrich Canada) and stored at -20 0 C until use. Flagellin was further purified of contaminant LPS by passage through polymixin B agarose (Detoxi-Gel, Pierce, Rockford, IL). A representative stained gel of purified and LPS-free flagellin is shown in Figure 4. Endotoxin-free flagellin was used for experiments on HEp-2 cells, U937 cells and THP-1 cells, but not on Caco-2 cells which are LPS-unresponsive. Flagellin preparation was considered Endotoxin-free if it did not induce significant amount of IL-8 secretion from 44 1 2 3 4 5 6 7 8 Fl iC-EAEC (65kDa) Figure 4: Purified FliC-EAEC: pjliC-EAEC was transformed into BLllpLysS. Log-phase culture containing polyhistidine tagged protein was purified by affinity chromatography, and proteins resolved by SDS-PAGE and gel stained with EZ blue. 1-Molecular weight standard, 2-8-Purified FliC-EAEC. 45 HEp-2 cells. In their native state, HEp-2 cells do not secrete IL-8 in response to purified FliC-EAEC. Therefore if a flagellin preparation was found to induce IL-8 secretion, it was considered to be due to contamination, and was not used for experiments on HEp-2, U937 and THP-1 cells. 2.6 Enzyme linked immunosorbent assay (ELISA) for measurement of IL- 8 secretion Due to the amplifying potential of enzyme labels, immunoassays that utilize enzyme-conjugated antibodies have become increasingly popular because of their high specificity and sensitivity [192]. In 1971, Engvall and Perlmann coined the term "enzyme-linked immunosorbent assay" which is perhaps better known by the acronym, "ELISA", to describe an enzyme-based immunoassay method, is useful for measuring antigen concentrations [193]. Cytokine Sandwich ELISA is a sensitive enzyme immunoassay that can specifically detect and quantify the concentration of soluble cytokine and chemokine proteins. The basic cytokine sandwich ELISA uses anti-cytokine antibodies (capture antibodies) that are coated onto plastic microwell plates. After plate washings, the immobilized antibodies serve to specifically capture soluble cytokine proteins present in the samples that were applied to the plate. After washing away unbound material, the captured cytokine proteins are detected by Biotin-conjugated anti-cytokine antibodies (detection antibodies) followed by enzyme labeled avidin or streptavidin. Following the addition of a chromogenic substrate-containing solution, the level of colored product generated by the bound enzyme can be conveniently measured spectrophotometrically using an ELISA plate reader at an appropriate wavelength. By including serial dilutions 46 of a standard cytokine protein of known concentration, a standard curve can be plotted using ELISA. Standard curves (or calibration curves) are generally plotted as the standard cytokine protein (typically pg or ng of cytokine/ml) versus the corresponding mean optical density (OD) values of replicates. The concentration of the putative cytokine-containing samples can be interpolated from the standard curve. To screen flagellin samples for IL-8 release, Caco-2 cells cultured in 24 well plates, were treated with FliC-E A E C protein samples (up to 10 ug in 50 ui). After 3 h, culture supernatant was tested for IL-8 concentration using a commercial kit to perform Sandwich ELISA (OptEIA kit, BD PharMingen, Mississauga, ON). Disposable plastic microwell strips (Removawell strips, V W R International) were coated with the capture antibody in Phosphate Buffered Saline (PBS) and 10% FBS, at a dilution of 1:250. The wells were incubated at 4 °C overnight and washed six times with PBST. The wells were blotted dry after every wash. Subsequently, 50 ul of sample (supernatant from cells treated with FliC-EAEC) or standard was added into the wells followed by detection antibody and enzyme concentrate, dissolved at 1:250 in PBS/10% FBS. The wells were incubated for 2-3 h at room temperature, and then washed six times again with PBST. Finally, 100 ul of chromogenic substrate solutions were added to the wells and allowed to incubate for 30 min at room temperature. The reaction was stopped by adding 50 ul of 2N H2SO4 and the plate was read at 450 nm-570 nm in an ELISA plate reader (Dynatech MR-5000, Dynatech Laboratories Inc., USA). 47 2.7 Pharmacological inhibitors of signaling proteins SB 203580, Bay 11-7082 and Wortmannin (Sigma Aldrich Canada) were dissolved in DMSO and stored in aliquots at -20 °C. On the day of the experiment, Caco-2 cells were fed with 500 ul of warm, fresh media in a 24 well plate, then treated with the inhibitor and incubated for 1 h at 37 °C. This was followed by the addition of 500 ng/ml of Fl iC-EAEC to the media and incubation for 3 h. At the end of this time point, culture supernatant was assayed for IL-8 by ELISA. Undifferentiated THP-1 cells cultured in 75 cm 2 culture flasks (VWR International) were washed with HBSS and adjusted to 106/ml in fresh media. THP-1 cells in 6 WP (2 X 106/well) were exposed to Fl iC-EAEC for 6 hrs at 37 °C. Supernatant were collected and clarified by centrifugation before measurement of IL-8 concentration by ELISA. 2.8 Western blotting and immunoprecipitation FliC-EAEC treatment of cells cultured in 6 well plates for various time points was followed by two washes with 2 ml of ice-cold Hanks balanced salt solution (HBSS) (Sigma Aldrich Canada). Treated cells were then exposed to 250-500 ul of lysis buffer (20 mM tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM E G T A , 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM P-Glycerol-phosphate, 1 mM Na3V04, 1 ug/ml leupeptin and 1 mM PMSF) on ice for 5-10 minutes and then scraped into microcentrifuge tubes using a rubber policeman. The tubes were then centrifuged to pellet debris and the supernatant transferred to another tube and stored at -80 °C until use for Western blotting and immunoprecipitation. 48 2.9 The determination of total cell lysate protein concentration The total cellular protein concentration from high speed supernatants were determined by the Lowry/DC assay (Bio-Rad Laboratories Ltd., Mississauga, Ont.) [194]. Bovine serum albumin (BSA) (Sigma Aldrich Canada) was used as standard for total cell protein concentration between 62.5 ug/ml and 4000 ug/ml protein dissolved in the above mentioned lysis buffer. Cleared cell lysate and serial dilutions of the BSA standards were loaded as 5 ul volumes into microwell strips. DC Lowry reagents were added and the reaction was allowed to develop for 15 minutes. The optical density for each standard and treated sample was read at 650 nm on an automated micro plate reader (model 550, Bio-Rad Laboratories Ltd., Mississauga, Ont.). Unknown protein concentration of the samples was determined from the BSA standard curve. 2.10 Polyacrylamide gel electrophoresis of cellular proteins Caco-2, THP-1 and HEp-2 proteins (50-100 \ig in clear cell lysate) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as first described by Laemmli [195]. Sample volumes corresponding to 50 ug of protein were aliquoted into microcentrifuge tubes with 4X loading buffer (187.5 mM Tris-HCl, pH 6.8, 30% glycerol, 6%SDS, 15% fi-mercaptoethanol) and were boiled for 5 minutes prior to loading onto a 4% stacking gel and 9% resolving gel in a Bio-Rad gel apparatus (Bio-Rad Laboratories Ltd., Mississauga, Ont.). Kaleidoscope molecular weight markers (Bio-Rad Laboratories Ltd., Mississauga, Ont.) were used to determine the mass of loaded proteins. Samples were run at for approximately one hour at 150 volts, or until the dye front reached the lower edge of the gel. 49 2.11 Western blotting for detection of cellular proteins by chemiluminescence Gels were disassembled from the gel apparatus and washed in deionized distilled water (ddFkO) for 5 min twice and then soaked in Towbin transfer buffer (48 m M Tris base, 39 m M glycine, 1.3 m M SDS, pH 9.2, 20% methanol) for 5 minutes. Proteins were transferred to 0.2 um polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories Ltd., Mississauga, Ont.) on a semi-dry electrophoretic transfer cell (Trans-Blot SD, Bio-Rad Laboratories Ltd., Mississauga, Ont.). India ink staining and appearance of the kaleidoscope colored molecular weight standards (Bio-Rad Laboratories Ltd., Mississauga, Ont.) on the PVDF membrane confirmed effective protein transfer. Blots were then blocked for one hour (in some experiments overnight) with 5% non-fat milk (3% BSA for blots involving the anti-phosphotyrosine antibody) dissolved in Tris buffered saline with 0.05% tween (TBST) (Tris 20 mM, NaCl 0.3 M , Tween-80, pH 7.4). Afterwards, the membranes were washed three times with TBST, and incubated in primary antibody for one hour at room temperature (in some experiments overnight at 4 °C). Primary antibodies were dissolved in TBST and used for Western blots as follows: rabbit anti-phospho-p38 M A P kinase and phospho-Akt primary antibodies (Cell Signaling technology, M A , USA) were used at a dilution of 1:500, rabbit anti-I-KB antibody (Santa Cruz biotechnology, CA, USA) was used at a dilution of 1:1000, mouse anti-IRAK (BD Biosciences, Mississauga, Ont.) at a dilution of 1:1000, mouse anti-p85a PI-3K (Upstate Biotechnology, M A , USA) at a dilution of 1:1000, and mouse anti-phosphotyrosine primary antibody, clone 4G10 (Upstate), at a dilution of 1:10,000. For immunoprecipitation, 1 ug/ml of 4G10 or rabbit anti-p38 M A P kinase was used per 100 5 0 ug of protein in cell lysate and incubated overnight at 4 °C by continuous agitation on a shaker. 50 ul of protein A-sepharose (Bio-Rad) was added to the sample in a centrifuge tube and incubated at 4 °C for 1 h on a rotating platform. Samples were then centrifuged to pellet the protein A-sepharose-antibody-antigen-complex, and the complex washed three times with cold lysis buffer, followed by three more washes with cold PBS. The beads were then resuspended in 4X gel loading buffer, boiled for 5 min and resolved in SDS-PAGE as indicated above. After incubation with the primary antibody, blots were washed with TBST three times for 10 min each, and then incubated with their corresponding horseradish peroxidase (HRP)-conjugated IgG secondary antibody for one hour at room temperature. Blots were then washed again three times for 10 min each and then exposed to enzymatic-enhanced chemiluminescence (ECL-plus) developing solution (Amersham Pharmacia Biotech, Quebec). Proteins were then detected on Kodak x-ray film (Fisher Scientific, Canada) film. Densitometry of the bands detected was performed using the Un-Scan-it software (Silk Scientific Inc., UT, USA). Background reading was subtracted before reading each band three times. Average values of each band were then used for statistical analysis using the student t-tests of significance, on Microsoft Excel software. Statistical analysis of all data in this thesis was perfomed by using the same test. 2.12 Generation of a mutant TLR5 Mutant TLR5 (Y798L) was generated by PCR-based in-vitro mutagenesis using double stranded DNA templates, and subsequently subjecting the PCR products to digestion with Dpnl restriction enzyme. In this method, two complimentary oligonucleotides are used to prime DNA synthesis by a high fidelity polymerase on a 51 denatured plasmid template. The two primers contain the desired mutation and have the same starting and ending positions on the opposite strands of the plasmid DNA. The entire length of both strands of plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutant plasmid containing staggered nicks on the opposite strands [196]. Methylated plasmid DNA is then digested with Dpn 1, reducing background transformants. The primers (Invitrogen Life technologies, Canada) for TLR5 mutagenesis were: Forward-ggtccttgtcccaattgcagttgatgaaa, reverse-tttcatcaactacaattgggacaaggacc. These primers were generated to introduce a mutation at amino acid position 798 of TLR5, by substituting a leucine for the tyrosine amino acid residue and introducing a Mun 1 restriction site for diagnostic digests. 50 ng of template plasmid- pEF6 (containing full length TLR5) was added to separate 45 pL aliquots of PCR reaction buffer to obtain a 50 ul reaction mixture containing Tris-HCl 10 mM (pH 8.3), KC1 50 mM, MgS0 4 3 mM, pooled dNTPs 100 u M , Pfu polymerase 2.5 units (MBI Fermentas) and 1 mM each of forward and reverse oligonucleotide primers (Invitrogen Lifetechnologies, Canada). 30 cycles of PCR were performed in a Delta Cycler-II (Ericomp, CA, USA) as follows: denaturation at 95 °C for 1 min, annealing at 44° C for 1 min and polymerization at 68° C for 17 minutes. Amplified PCR products were resolved on 0.9% agarose gel containing 0.5 ug/ml of ethidium bromide, and a band corresponding to 8.4 kb was identified under U V light. 10 ul of amplified DNA was digested with 10 units of Dpnl at 37 °C for 1 hour. The digested products were resolved on 0.9% agarose gel and visualized under U V light as above. The mutant TLR5 DNA was later prepared by transforming competent E. coli, HB 101, with luL of digested DNA according to method outlined above and extraction 52 by Plasmid Midikit (Qiagen Inc., Mississauga, Ontario). The 798 Y—»L mutation of TLR5 was confirmed by sequencing at the Nucleotide and Protein sequencing (NAPS) unit, U B C . 2.13 In-vitro enzyme assay for measurement of PI-3K activity An in-vitro kinase assay was used to measure the activity of PI-3K in FliC-EAEC-treated Caco-2 cells [197]. Aliquots of cell lysates adjusted for protein content (300-500 ug of protein) were incubated overnight at 4 °C with 5 ul mouse monoclonal antibody to p85a subunit of PI-3K, and immune complexes were adsorbed onto protein A-agarose (Bio-Rad Laboratories Ltd., Mississauga, Ont.) for 1 h at 4°C. The complexes were washed twice with lysis buffer and three times with Tris-HCl 10 m M (pH 7.4). Phosphatidylinositol (PI) was dissolved in 30 m M HEPES (Sigma Aldrich Canada) and sonicated (3 x 20 s) in Sonic Dismembrator 60 Sonicator (Fisher Scientific). 10 ul of PI was added to 40 ul of kinase buffer (Hepes 30 mM, MgCb 30 mM, adenosine 200 uM, ATP 50 UM , and 10 uCi of y 3 2 P-ATP). Kinase buffer was added to immune complexes in 1.7 ml centrifuge tubes and reactions were carried out for 15 min at room temperature. The reaction was stopped by the addition of 100 ul of 1 N HC1 and 200 ul of chloroform : methanol (1:1, v/v) (Fisher Scientific Canada). Lipids were separated on oxalate-treated silica TLC (VWR International) plates using a solvent system of chloroform : methanol : water : 28% ammonia (45:35:7.5:2.5, v/v/v/v). Plates were exposed to Kodak x-ray film at -70 °C. Incorporation of radioactivity into lipids was quantified by excising the corresponding portions of the TLC plate followed by liquid scintillation counting. 53 Chapter III: Results 3.1 Result: FliC-EAEC activates Akt and degrades I-KB in Caco-2 cells PI-3K activation was determined by in-vitro kinase assay in Caco-2 cells treated with F l iC-EAEC. This method is very sensitive in detection of enzyme activity because the kinase reaction involves y- 3 2 P-ATP, making it possible to detect and quantify the signals by densitometry [198]. Results shown in Figure 5 are representative of data from this experiment (n = 7). PI-3K activity was studied from 1 min to 10 min of incubation with F l iC-EAEC (500 ng/ml) (Figure 5A). Although a two-fold activation of PI-3K was noted at 3 min of incubation, repeated experiments did not demonstrate a statistically significant increase of activity. IGF-II (5 ng/ml) treatment of Caco-2 cells for 5 min, in presence and absence of Wortmannin, was also performed but failed to activate PI-3K (Figure 5B). Since PI-3K undergoes tyrosine phosphorylation upon activation, a mouse monoclonal antiphosphotyrosine antibody (4G10) was used to immunoprecipitate PI-3K from Fl iC-EAEC treated Caco-2 cell lysates. Protein A-sepharose adsorbed proteins were resolved on SDS-PAGE, transferred to PVDF membranes and the blots probed with a mouse monoclonal anti-p85oc PI-3K antibody. The results of this experiment are shown in Figure 6. Increased phosphorylation of PI-3K was seen at 10 min of incubation with F l iC-EAEC, by detection of a band at 85 kDa. This was blocked by 1 hour pre-treatment with Wortmannin (100 nM). Equal loading of proteins in all lanes was confirmed by staining with India ink. 54 FliC A) Density Units Mean + SEM n=7 Control V 10 nun 140 i 120 -J 100 8 0 6 0 -4 0 2 0 -0 I I I B) Control WM FliC FliC IGF IGF + + WM WM Figure 5: Fl iC-EAEC activation of PI-3K in Caco-2 cells: Equal amounts of protein (500 pg) from cell lysates were incubated with anti-mouse primary p85 PI-3K antibody overnight and PI-3K activity measured by in-vitro kinase assay. A) Time-course of incubation due to FliC-EAEC (500 ng/ml). Bands were analyzed by densitometry. B) Effects of Wortmannin on FliC-EAEC and IGF-II induced activation measured by in-vitro kinase assay. 1 h pre-incubation with Wortmannin (100 nM) was followed by 5 min exposure of Caco-2 cells to Fl iC-EAEC (500 ng/ml) and IGF-II (5 ng/ml). 55 PI-3K (85kDa)-Control WM FliC WM FliC WM (3 min) + (10 min) + FliC FHC (3 min) (10 min) Density Units Mean n=2 100 i 90 -80 -70 60 50 •40 30 -20 -10 -0 Control FliC (10 min) Figure 6: FliC-EAEC-induced PI-3K tyrosine phospho-activation. Caco-2 cells were pre-incubated with Wortmannin (100 nM) for 1 h and exposed to Fl iC-EAEC (500 ng/ml) for 3 and 10 min. Cell lysate containing 500 )ig of protein/sample was used for immunoprecipitation with a mouse monoclonal anti-phosphotyrosine antibody (4G10) overnight, then analyzed by Western blotting using a mouse anti-p85-PI-3K primary antibody. Differences were analyzed by densitometry. Equal loading of proteins was confirmed by India ink staining of membranes. 56 Another approach to study PI-3K involvement due to F l iC-EAEC is to examine the activation of a downstream target of PI-3K activation such as Akt (PKB). A rabbit monoclonal anti-phospho-Akt primary antibody that recognizes phosphorylation of Akt at serine 473 was used for this purpose. A Western blot of time-course of Akt activation due to F l iC-EAEC (500 ng/ml) is shown in Figure 7A. Akt activation was detected by identifying a band at 60 kDa beginning at 20 min of incubation with F l iC-EAEC, reaching its maximum at 60 min and subsiding by 120 min to near baseline levels. Activation of Akt was found to be statistically significant at 60 min incubation with FliC-E A E C . Increasing amounts of F l iC-EAEC (0.2 ng/ml to 3125 ng/ml) activated Akt in Caco-2 cells after 60 min of incubation in a dose-dependent manner (Figure 7B). Pre-incubation with Wortmannin (100 nM) for 1 hr inhibited activation of Akt (Figure 7C). One downstream effect of PI-3K activation is I-KB degradation, releasing the transcription factor N F - K B to migrate to the nucleus. Degradation of I-KB due to FliC-E A E C (500 ng/ml) was studied by Western analysis using a rabbit polyclonal anti-I-KB primary antibody. Figure 8A shows a time-course of I-KB degradation beginning at 10 min of treatment of Caco-2 cells with 500 ng/ml of F l iC-EAEC, by loss of the band intensity at 37 kDa. This was restored to its normal levels by 60 min of incubation. In the presence of 100 nM Wortmannin, F l iC-EAEC did not degrade I-KB (Figure 8B), suggesting that PI-3K activity is required for I-KB degradation in response to Fl iC-EAEC. To determine if PI-3K activation was essential in F l iC-EAEC induced IL-8 secretion, U937 leukemic cells with a mutation (see Materials and Methods) in the p85 regulatory subunit of PI-3K (DN) were treated with F l iC-EAEC, and IL-8 secretion compared with wild type (WT) U937 cells. As shown in Figure 9, the amount of IL-8 57 A) Phospho-Akt (60kDa) > - m Control 10 20 30 40 60 120 min 100 -I 80 -Density Units 60 • Mean + SEM n=4 4 0 20 0 Control FliC (60min) *p< 0.001 FliC (ng/ml) Control 0.2 1 5 25 125 625 3125 C) PhospnoAkt (60kDa) Control WM FliC WM + FliC Figure 7: Phospho-activation of Akt by Fl iC-EAEC in Caco-2 cells. 50p,g/sample of protein from cell lysate was analyzed by Western blots using a rabbit anti-phopho-Akt primary antibody. A) Time-course of Fl iC-EAEC (500 ng/ml) induced activation of Akt. Densitometry was performed on signals from untreated control and F l iC-EAEC (60 min) stimulated samples. B) Dose-response of FliC-EAEC-induced activation for 60 min. Results are representative of two independent experiments. C) Effect of Wortmannin (100 nM) pre-incubation (lhr) on Akt activation. Results are representative of three independent experiments. Equal loading of proteins was confirmed by India ink staining of membranes. 58 A) I-KBa(37kDa) Control 10 30 60 120 nun. B) I-KBrx(37kDa) Control W M FliC W M + FliC 1 2 0 -• 1 0 0 -Density Units 8 0 -Mean + SEM 6 0 n=3 4 0 2 0 -J 0 Control FliC 30 min *p<0.05-^Control Figure 8: 1-KBCC degradation by Fl iC-EAEC in Caco-2 cells. Equal amounts of proteins (50 pg/sample) from cell lysates were subjected to Western analysis using a rabbit anti-I-KB primary antibody. A) Time-course of I-KBCC degradation by FliC-E A E C (500 ng/ml). B) Effect of 1 h pre-treatment with Wortmannin (100 nM) on I-KBCC degradation by F l iC-EAEC (500 ng/ml) incubation for 30 min. Equal loading of proteins was confirmed by India ink staining of membranes. 59 released from D N U937 cells was not significantly different from that of WT U937, when treated with equal amounts of Fl iC-EAEC (500 ng/ml). In contrast, LPS (10 ug/ml) induced IL-8 secretion was markedly blunted from U937 cells containing D N p85 as compared to WT p85 U937 cells (p<0.05). This data suggests that IL-8 secretion in U937 cells due to Fl iC-EAEC is not affected by mutation of p85 subunit of PI-3K. To elucidate the mechanisms of IL-8 release from Caco-2 cells exposed to FliC-E A E C , we studied the effects of pharmacological inhibitors on IL-8 secretion. These inhibitors were used at lowest concentration possible to avoid non-specificity of their action. Wortmannin (PI-3K inhibitor) was used at concentration of 100 nM for inhibition of IL-8 secretion from Caco-2 cells. This dose is effective for inhibition of PI-3K, as higher concentrations (300 nM) have been shown to inhibit myosin light chain kinase (MLCK) [199]. Bay 11-7082 (a N F - K B inhibitor) has an IC50 of 10 uM [200] and was used at a concentration of 20 uM for all experiments in this thesis. Inhibition of IL-8 secretion was studied by incubating cells with the inhibitors for 1 hour, followed by exposure to Fl iC-EAEC or a positive control such as IL-1 p. Figure 10 shows the effects of Wortmannin and Bayl 1-7082 on Fl iC-EAEC-induced IL-8 release from Caco-2 cells. Fl iC-EAEC (500 ng/ml) and 10 ng/ml of IL-1 p caused 966+156 pg/ml and 1763+194.67 pg/ml of IL-8 secretion respectively after 3 hrs. Pre-incubation with Bay 11-7082 (20 uM) produced a 30% decrease in IL-8 release, whereas 100 nM Wortmannin had no effect. The presence of both Bay 11 and Wortmannin resulted in 40% inhibition of the maximal Fl iC-EAEC induced IL-8 release. 60 IL-8 pg/ml Mean + SEM n=3 1400 i 1200 -1000 -800 600 -400 -200 0 Control FliC WTp85 DNp85 LPS *p<0.05vs WTp85-PI-3K Figure 9: IL-8 secretion from WT and DN p85-PI-3K U937 cells. U937 cells (2 X 106) with WT and mutant PI-3K were exposed to FliC-EAEC (500 ng/ml) in 6WP for 6 h. Supernatants were analyzed for IL-8 concentration by ELISA. 61 2500 -, 2000 1500 IL-8 pg/ml Mean + SEM n=3 1000 500 Control FliC B + FliC C-Control B-Bayl l WM-Wortmaiuiin *p < 0.05 vs FliC ** p <0.05vs IL-lp Figure 10: Effect of N F -KB and PI-3K inhibition on FliC-EAEC-induced IL-8 release from Caco-2 cells. Pre-treatment of Caco-2 cells with Bay 11 (20 uM) and Wortmannin (100 nM) for 1 h, was followed by incubation with Fl iC-EAEC (500 ng/ml) and IL-lp (20 ng/ml) for 3h. 6 2 Similarly, Bay 11 inhibited IL-1 B induced IL-8 secretion by 40% whereas Wortmannin had no effect. Our data suggests that although secretion of IL-8 is unaffected by PI-3K inhibition, NF-KB inhibition decreases IL-8 secretion from Caco-2 cells upon treatment with F l iC-EAEC. 3.2 Result: FliC-EAEC activates p38 MAP kinase in Caco-2 cells To determine whether p38 M A P kinase is involved in FliC-EAEC-induced IL-8 secretion, SB 203580 was tested in Caco-2 cells. As shown in Figure 11, when Caco-2 cells were incubated with 10 uM SB 203580 alone for 1 hour and exposed to F l iC-EAEC for 3 hours, IL-8 secretion decreased by 90% (p<0.05). In the presence of SB 203580 and Bay 11, IL-8 secretion was reduced to levels almost identical to untreated control samples. A similar trend was observed with IL-1 B (20 ng/ml), where pretreatment with 10 uM SB 203580 resulted in 90% inhibition of IL-8 and 20 u M Bay 11 caused approximately 50% inhibition. As noted with F l iC-EAEC, the combination of both inhibitors reduced IL-8 release in response to IL-ip, approximately equal to untreated control samples. DMSO was used as a vehicle for SB 203580 and for other inhibitors (Wortmannin and Bay 11). SB 203580 (10 uM) alone did not release IL-8 in Caco-2 cells and was not toxic to cells. This was confirmed by trypan blue staining of Caco-2 cells after 6 hrs of treatment with the inhibitor. Two concentrations of SB 203580 were tested to determine the dose that would cause 50% inhibition of F l iC-EAEC induced IL-8 secretion from Caco-2 cells (Figure 12). 3 uM concentration caused approximately 50% inhibition (p<0.05) of 100 ng/ml and 1000 ng/ml F l iC-EAEC induced IL-8 secretion, and 90% inhibition of F l iC-EAEC was seen with 10 uM SB 203580 (p<0.05). The level of inhibition in this experiment is 63 consistent with the data from experiments performed earlier (Figure 11) where both SB 203580 and Bay 11 reduced IL-8 secretion. Activation of p38 M A P kinase was also studied by examining the characteristics of its phosphorylation. Caco-2 cell lysates treated for 30 min with F l iC-EAEC (500 ng/ml), were immunoprecipitated with p38 M A P kinase antibody overnight and subjected to Western blots for tyrosine phosphorylation. The results of this experiment are shown in Figure 13A. Exposure to F l iC-EAEC for 1 h induced tyrosine phosphorylation of p38 M A P kinase when compared to untreated control samples. Figure 13B shows phosphorylation of p38 M A P kinase by Fl iC-EAEC after 30 min of incubation, detected with an antibody that recognizes dual phosphorylation of p38 M A P kinase at tyrosine and threonine residues. Treatment of Caco-2 cells with IL-1 P (20 ng/ml) caused a similar activation and was included as a positive control in all p38 M A P kinase experiments. A time-course of activation of p38 M A P kinase in Caco-2 cells confirmed that maximal activation of p38 M A P kinase occurred at 30 min of incubation with 500 ng/ml of F l iC-EAEC (Figure 14A). Densitometry showed that the phospho-p38 M A P kinase signal at 30 min was approximately ten-fold higher than the untreated control sample (pO.OOl). Figure 14B shows that all samples contained equal quantities of total p38 M A P kinase in the cell lysate. The effect of SB 203580 treatment on p38 M A P kinase phospho-activation due to F l iC-EAEC was examined by Western blots (Figure 15). The data showed that SB 203580 (10 uM) inhibited F l iC-EAEC (500 ng/ml) and IL- lp (20 ng/ml) induced phospho-activation, observed by the loss of the p38 M A P kinase band at 43 kDa. This experiment was performed once. 64 2000 1800 1600 -1400 -IL-8 p^ml 1 2 0 0 Mean + SEM n=3 1000 800 600 400 200 -| 0 SB FL If. *p< 0.05 vs FliC **p<0.05\sIL-10 V-DMSO SB 203580-SB Bay 11-B i SB + EL 1 B SB + + EL 1 B + EL 1 Figure 11: Effect of p38 MAP kinase and NF-KB inhibition on FliC-EAEC-induced IL-8 release from Caco-2 cells. Cells pre-treated for 1 h with SB 203580 (10 uM) and Bay 11 (20 uM) were exposed to FliC-EAEC (500 ng/ml) or IL-1 (3 (20 ng/ml) for 3 h. 65 N o S B S B 3uM — * — S B lOuM 1 6 0 0 i 0.1 1 10 100 1 0 0 0 FliC ng/ml * p <0.05 vsNo SB Figure 12: Effect of p38 MAP kinase inhibitor on IL-8 secretion from Caco-2 cells incubated with Fl iC-EAEC. Pre-incubation of Caco-2 cells with varying doses of SB 203580 (SB) for 1 h was followed by exposure to indicated concentrations of FliC-E A E C for 3 h. 66 A) phospho-p38 MAP kinase (43kDa) 100 SO Density Units Mean + SEM n=3 60 43 20 C FliC IP- p38MAP kinase WB- anti-phosphotyrosine (4G10) Control FliC *p<0.05w Control B) Phospho-p38 MAP kinase (43kDa) Control FliC 30 min IL-1 30 min Figure 13: Phospho-activation of p38 MAP kinase by Fl iC-EAEC in Caco-2 cells. A) Caco-2 cells were treated with FliC-EAEC (500 ng/ml) for 30 min. Cell lysates (500 p.g protein/sample) were incubated with rabbit phospho-p38 M A P kinase primary antibody overnight, resolved by SDS-PAGE and probed with mouse primary anti-phosphotyrosine antibody (4G10) in western blots. B) Caco-2 cells were incubated with Fl iC-EAEC (500 ng/ml) or IL-lp (20 ng/ml) for 30 min. 50 u.g of cell lysate was analyzed by SDS-PAGE and phospho-p38 MAP kinase antibody in Western blots. Results are representative of 3 independent experiments. Equal loading of protein samples was confirmed by India ink staining of membranes. 67 A ) Phospho-p38 B) Total-p38 15 ruin FliC 30 min 60 nun — % IL-lp 120 10 min min 43kDa • 43kDa Density Units Mean + S E M n=4 loo -9 0 -8 0 -7 0 -6 0 5 0 4 0 3 0 2 0 1 1 0 -I 0 * pO.001 FliC 30 min Figure 14: Time-course of p38 MAP kinase phospho-activation by Fl iC-EAEC. A) Caco-2 cells were incubated with FliC-EAEC (500 ng/ml) or IL-lp (20 ng/ml) for time points indicated. Equal amounts of protein (50 p.g/sample) were analyzed by Western blots using a rabbit anti-phospho-p38 MAP kinase primary antibody. B) Blots were stripped and re-probed with rabbit p38 MAP kinase primary antibody. Untreated control and Fl iC-EAEC stimulated (30 min) samples were analyzed by densitometry. 68 P h o s p h o - p 3 8 M A P k inase ( 4 3 k D a ) C SB SB FliC SB IL-1 + + FliC IL-1 Figure 15: Inhibition of FliC-EAEC-induced p38 MAP kinase phospho-activation. Caco-2 cells were pre-incubated with SB 203580 (10 uM) for 1 h and exposed to Fl iC-EAEC (500 ng/ml) or IL-lp (20 ng/ml) for 1 h Equal amount of protein (50 ug/sample) from cell lysate were subjected to Western analysis using rabbit anti-phospho-p38 MAP kinase primary antibody. Equal loading of proteins was confirmed by India ink staining of membranes. 69 3.3 Result: FliC-EAEC causes IL-8 secretion, Akt and p38 MAP kinase activation in THP-1 cells. Flagellin induced secretion of IL-8 by THP-1 cells has not been demonstrated previously. Therefore, experiments were performed to determine i f F l iC-EAEC treated THP-1 cells would release IL-8. A time-course of F l iC-EAEC induced IL-8 secretion from undifferentiated THP-1 cells was performed to determine the time point at which maximal IL-8 secretion occurs in this cell line. THP-1 cells (2 X 106 cells) were incubated with F l iC-EAEC (500 ng/ml) for 0, 0.5, 1, 3, 6 and 12 hrs (Figure 16). The amount of IL-8 secretion increased rapidly between 3-6 hours, reaching a plateau at 12 hrs of incubation with F l iC-EAEC. Since sub-maximal IL-8 secretion was measured at 6 hours (1065.35+39.4 pg/ml), this time point was selected for subsequent experiments. A dose-response in THP-1 cells indicated that (Figure 17) F l iC-EAEC, between 250-1500 ng/ml, caused a dose-dependent increase in IL-8 secretion after 6 hrs of incubation. Although significant IL-8 secretion release was measured due to 250 ng/ml of Fl iC-EAEC, maximal IL-8 secretion was seen at 1500 ng/ml. For studies of signaling pathways, a concentration of 500 ng/ml of F l iC-EAEC was used in experiments below. The effect of PI-3K and p38 M A P kinase inhibition on FliC-EAEC-induced IL-8 secretion was evaluated in THP-1 cells. Exposure to F l iC-EAEC (500 ng/ml) caused significant amount of IL-8 secretion. Pre-incubation of THP-1 cells with SB 203580 (10 pM) caused 50% reduction of the maximal FliC-EAEC-induced IL-8 secretion (1417.3 + 57.7 pg/ml to 756.5 ± 41.2 pg/ml) (p<0.05) (Figure 18). In contrast, Wortmannin (100 nM) did not inhibit IL-8 secretion from THP-1 cells exposed to F l iC-EAEC. Similarly, 70 SB 203580 decreased LPS-induced IL-8 secretion, whereas Wortmannin did not cause a reduction. The result of an in-vitro kinase assay for activation of PI-3K in THP-1 whole cell lysates is shown in Figure 19. 5 min incubation of THP-1 cells with 500 ng/ml of Fl iC-E A C did not activate PI-3K (19A), whereas incubation with LPS (10 p.g/ml) caused a three-fold activation. Akt activation was also studied using a rabbit anti-phospho Akt antibody. Identification of a band at 60 kDa indicated phospho-activation due to 500 ng/ml of F l iC-EAC that was maximal after 60 min of incubation. LPS (10 |a.g/ml) was included as a positive control in this experiment (19B). The activation of p38 M A P kinase in response to F l iC-EAEC in THP-1 cells was studied by a phospho-p38 M A P kinase antibody in Western blots. A shown in Figure 20, 500 ng/ml of F l iC-EAEC was added to 2 X 106 undifferentiated THP-1 cells in a volume of 2 ml in 6WP. Activation of p38 M A P kinase was studied at 1 and 2 hr of incubation with F l iC-EAEC in the whole cell lysates of THP-1 cells and compared to that of the untreated controls. Treatment of THP-1 with LPS (10 ug/ml) for 1 h was included as positive control. A ten-fold activation of p38 M A P kinase (p < 0.01) was seen in THP-1 cells incubated with F l iC-EAEC for 2 hrs. LPS also caused activation after 1 hr of incubation. 3.4 Result: FliC-EAEC treatment of HEp-2 cells transiently expressing TLR5 causes p38 MAP kinase-dependent IL-8 secretion and IRAK activation. The roles of p38 M A P kinase and PI-3K were studied in TLR5-transfected HEp-2 cells incubated with Fl iC-EAEC. After 48 hours of TLR5 transfection, HEp-2 cells secre-71 1600 - i 1400 -200 -0 4 1 r 1 1 1 r 1 0 2 4 6 8 10 12 14 Time (hrs) Figure 16: Time-course of FliC-EAEC-induced IL-8 secretion from THP-1 cells. Undifferentiated THP-1 cells (2 X 106/well) in 6 well-plates were incubated with FliC-E A E C (500 ng/ml) for time points indicated. IL-8 secretion was measured in supernatant by ELISA. 72 0 500 1000 1500 2000 2500 FliC (ng/ml) Figure 17: Dose-response of FliC-EAEC-induced IL-8 secretion from THP-1 cells. Undifferentiated THP-1 cells (2 X 106/well) in 6 well-plates were incubated for 6 h with increasing doses Fl iC-EAEC as indicated. IL-8 secretion in the supernatant was quantified by ELISA. 73 2500 -, C SB WM F SB WM SB LPS S B WM S B + + + + + + F F WM LPS LPS WM + + F LPS * p < 0.05 vs C ** p < 0.05 vs FliC *** p < 0.05 vs LPS Figure 18: Inhibition of IL-8 secretion from THP-1 cells exposed to Fl iC-EAEC. THP-1 cells (2 X 106/sample) in 6 well-plates were pre-incubated with SB 203580 (SB-10 uM) and Wortmannin (WM-100 nM) for 1 h and then exposed to Fl iC-EAEC (500 ng/ml) and LPS (10 ug/ml) for 6 h. Secretion of IL-8 in supernatants was measured by ELISA. 74 C FliC LPS A) PI-3K (in-vitro kinase assay) B) Phospho-Akt (fjOkDa) » * « ~ » - r 3 » ,* Control FliC FliC LPS 30niin 60roin 60rnin Figure 19: Activation of Akt by FliC-EAEC in THP-1 cells. A) Undifferentiated THP-1 cells (2 X 106/sample) were treated with Fl iC-EAEC (500 ng/ml) for 5 min in 6 well-plates. Equal amounts of protein (500 ug) from cell lysates were incubated with mouse monoclonal anti-p85 PI-3K antibody overnight and activity of PI-3K measured by in-vitro kinase assay. Result representative of three independent experiments. B) THP-1 cells were incubated with Fl iC-EAEC (500 ng/ml) and LPS 10 (pg/ml) for the time points indicated. Equal amounts of protein (50 ug/sample) from cell lysate were subjected to Western analysis using a rabbit anti-phospho-Akt primary antibody. Result representative of three independent experiments. Equal loading of proteins was confirmed by India ink staining of membranes. 75 Phospho-p38 MAP kinase (43kDa) " Control FliC 1 h IS LPS Density Units Mean + SEM n=3 120 100 SO 60 40 20 0 Control *;p;.<0.00lvs Control FliC 2 h Figure 20: F l iC-EAEC activates p38 M A P kinase in THP-1 cells. Undifferentiated THP-1 cells (2 X 106/sample) were incubated with F l iC-EAEC (500 ng/ml) and LPS (10 ug/ml) for the time points indicated. Equal amounts of protein (50 ug) from cell lysates were analyzed by Western blots using rabbit anti-phospho-p38 M A P kinase primary antibody. Equal loading of proteins was confirmed by India ink staining of membranes. Bands were compared by densitometry. 76 -ted significantly higher quantities of IL-8 after F l iC-EAEC (500 ng/ml) treatment for 3 hr (p < 0.05), than untransfected cells and untreated TLR5-transfected cells (Figure 21). Inhibition of p38 M A P kinase with SB 203580 led to a significant reduction of Fl iC-EAEC-induced IL-8 release (p < 0.05) (Figure 22). The concentration of IL-8 measured in supernatants from HEp-2 cells incubated with SB 203580 alone was similar to untransfected, control samples. An antibody that recognizes phosphorylated p38 M A P kinase in Western blots was used to measure activation of p38 M A P kinase. As shown in Figure 23, 1 hr treatment with F l iC-EAEC (500 ng/ml) caused an approximately three-fold increase in the band detected at 43 kDa from TLR5-transfected HEp-2 cells (p < 0.05). Treatment with IL-1 (3 (20 ng/ml) also caused activation of p38 M A P kinase, as others have shown [201]. The effect of F l iC-EAEC on HEp-2 cells transfected with TLR5 containing a mutation (Y798L) in a putative PI-3K binding domain (AT5), was also studied (Figure 24). FliC-EAEC-induced IL-8 release from HEp-2 cells transfected with WT TLR5 (WT5) was approximately three-fold higher than from untreated TLR5 transfected cells. Further, incubation of F l iC-EAEC with AT5 transfected cells did not increase IL-8 secretion, and the levels were comparable to untreated AT5 HEp-2 cells. A Western blot showed that phospho-activation of p38 M A P kinase was greater in HEp-2 cells transfected with WT5 than with AT5 transfection (Figure 25). Effects of F l iC-EAEC on IRAK were studied by Western blot using a primary anti-rabbit antibody, which recognizes the phosphorylated form of this enzyme in Caco-2 and TLR5-transfected HEp-2 cell lysates. Incubation of Caco-2 cells for 15 min with 77 Fl iC-EAEC (500 ng/ml) and IL-lp (20 ng/ml) for 10 min, resulted in increased phosphorylation of IRAK, shown in Figure 26, where an intense band of 100 kDa was detected. After 2 hrs of stimulation with Fl iC-EAEC, there was approximately a 50% reduction of the IRAK signal. Similarly, I R A K activation was seen in TLR5-transfected HEp-2 cells after FliC-E A E C (500 ng/ml) exposure for 15 min (Figure 27). Compared to untreated TLR5-transfected cells, a three-fold increase of IRAK activation was noted in TLR5-transfected HEp-2 cells treated with Fl iC-EAEC. Activation was also seen in untransfected HEp-2 exposed to IL-ip (20 ng/ml) for 15 min. 78 1400 _ 1200 J 1000 -I IL-8 pg/ml goo Mean + SEM n=4 600 J 400 J 200 UT UT + FliC T GFP GFP + FliC T5 + Media T5 + FliC * p < 0.05 vs TLR5 + Media Figure 21: F l iC-EAEC induction of IL-8 secretion from HEp-2 cells transiently transfected with TLR5. HEp-2 cells co-transfected with GFP and TLR5 for 48 h were incubated with F l iC-EAEC (500 ng/ml) for 3 h. IL-8 concentration in the supernatant was measured by ELISA and shown as above. 79 1800 1600 1400 1200 IOOO H IL-8 pg/ml Mean + S E M 800 A n=3 600 H 400 200 H 0 UT UT + SB UT + FliC T5 + Media *p<0.05\sT5 + Media ** p<0.05\BT5+ Fl iC T5 + Fl iC T5 + SB + Fl iC Figure 22: Inhibition of FliC-EAEC-induced IL-8 secretion from TLR5-transfected HEp-2 cells. Untransfected and TLR5-transfected HEp-2 cells were pre-incubated with SB 203580 (10 uM) for 1 h and then treated with Fl iC-EAEC (500 ng/ml) for 3 h. IL-8 was measured by ELISA. 80 Phospho-p38 M A P kinase (43kDa) Control 100 -, 8 0 Density Units 6 0 - | n=4 4 0 -2 0 -0 Control TLFv.5 TLR5 + FliC TLR5 TLR5 + FliC * p < r j . 0 5 \ 3 T L R 5 * * p < 0 . 0 5 vs Control IL-lp T IL-lp Figure 23: Activation of p38 M A P kinase in TLR5-transfected HEp-2 cells exposed to F l i C - E A E C . Transiently transfected HEp-2 cells were incubated with F l i C - E A E C (500 ng/ml) or IL-1 p (20 ng/ml) for 1 h and were analyzed by Western blots with rabbit anti-phospho-p38 M A P kinase antibody. Signals were compared by densitometry. 81 600 -, 500 400 • IL-8 pg/ml Mean + SEM 300 n=2 200 -100 -WT5 WT5 + FliC AT5 AT5 + FliC Figure 24: FliC-EAEC induction of IL-8 release from HEp-2 cells transfected with wild type (WT5) and Y798L TLR5 (AT5). Transiently transfected HEp-2 cells were incubated with FliC-EAEC (500 ng/ml) for 3 h and IL-8 concentration in supernatants measured by ELISA. 82 Phospho-p38 MAP kinase (43kDa) T 5 T 5 A T 5 A T 5 + + FliC FliC Figure 25: Activation of p38 MAP kinase by Fl iC-EAEC in HEp-2 cells transfected with wild type (WT) and Y798L TLR5 (AT5). Transfected cells were incubated with FliC-EAEC (500 ng/ml) for 1 h and cell lysate (50 ug/sample) was analyzed by Western blots using a rabbit anti-phospho-p38 MAP kinase antibody. 8 3 Phospho-IRAK (100 kDa) Control 15 min FliC 1 hr 2hr IL-lp 10 min 100-I 80-Density Units ffl "I n = 4 40 20 H 0 Control FliC 15 min * p < 0.05 Figure 26: Activation of IRAK by FliC-EAEC in Caco-2 cells. Cells were treated with Fl iC-EAEC (500 ng/ml) or IL-ip (20 ng/ml) for times indicated. Equal amounts of protein (50 ug/sample) were studied by Western analysis using mouse anti-phospho-IRAK antibody. Bands were compared after densitometry. Equal loading of proteins was confirmed by India ink staining of membranes. 84 Phospho-IRAK (lOOkDa) UT T5 T5 + FliC IL-lp 150 -, 100 A Density Units n=4 50 A 0 T5 + FliC * p<0.05 Figure 27: IRAK activation in TLR5-transfected HEp-2 cells treated with Fl iC-EAEC. Transfected cells were exposed to FliC-EAEC (500 ng/ml) or IL-lp (20 ng/ml) for 15 min. Cell lysate (50 ug protein/sample) were studied by Western blot using mouse anti-phospho-IRAK primary antibody. Densitometry was performed for comparison of bands. Equal loading of proteins was confirmed by India ink staining of membranes. 85 Chapter IV: Discussion 4.1 Effects of FliC-EAEC on PI-3K signaling pathways in Caco-2 cells The results of the series of experiments on Caco-2 cells suggest that PI-3K dependent pathways are not required in F l iC-EAEC induced IL-8 release, as we were unable to conclusively demonstrate activation of PI-3K in Caco-2 cells (Figure 5). Lack of a positive control for induction of PI-3K activity in Caco-2 cells made it difficult to confirm our findings. A n inducer of PI-3K has not been previously reported in Caco-2 cells; however, Insulin-like growth factor (IGF-II) receptors are expressed in Caco-2 cells [202]. When Caco-2 cells were stimulated with IGF-II, PI-3K activation was not detected. As phosphorylation of PI-3K is an essential for its activation, analysis of immunoprecipitated proteins showed that 10 min stimulation with F l iC-EAEC caused tyrosine phosphorylation of p85cc subunit of PI-3K (Figure 6) in Caco-2 cells, which was inhibited by Wortmannin. Another approach to studying the activity of an enzyme is to measure its downstream target, which is an indirect means of studying the upstream mediator. Akt (PKJ3) is an important signaling protein functioning distal to PI-3K, and is involved in several of its cellular effects such as cell survival pathways, the regulation of gene expression and cell metabolism [203]. F l iC-EAEC activation of Akt is suggestive of PI-3K activity upstream (Figure 7), which we failed to detect. This may be attributed to a constitutive PI-3K activity and lack of positive control in Caco-2 cells. Time-course experiments show that phospho-activation of Akt occurs early (20 min) during FliC-E A E C treatment, and is maximal at 60 min with approximately ten-fold increase in the signal. The involvement of PI-3K signaling is supported by the dose-dependent 86 activation of Akt and importantly, inhibition by Wortmannin. In some experiments, a high phospho-Akt signal from the untreated control sample suggests basal Akt activity. Another consequence of PI-3K signaling is I-KB degradation and N F - K B nuclear migration. As expected, (Figure 8) F l iC-EAEC treatment caused I-KB degradation, which was reversible by Wortmannin, again implicating the PI-3K/Akt pathway. Although Akt is activated by Fl iC-EAEC, IL-8 secretion does not seem to require PI-3K. U937 cells containing a mutation in the p85 regulatory subunit of PI-3K did not affect IL-8 secretion due to F l iC-EAEC in U937 cells (Figure 9). Moreover, pharmacological inhibition of PI-3K did not decrease IL-8 release, whereas a N F - K B inhibitor caused a significant decrease (Figure 10), suggesting that N F - K B activation is independent of PI-3K or a redundant mechanism exists. Collectively, these data suggest that F l iC-EAEC causes phospho-activation of Akt and I-KB degradation in Caco-2 cells. Wortmannin pre-treatment indicates that these effects are a consequence of PI-3K activity, although this is not necessary for IL-8 secretion. Moreover, experiments with Bay 11-7082 revealed that 1-KB/NF-KB system partially contributes to IL-8 release, raising the possibility that N F - K B involvement in this pathway could be due to another separate pathway besides PI-3K. The biological significance of Akt activation by F l iC-EAEC is presently unknown. Because Akt was activated after 20 min incubation with F l iC-EAEC, it is unlikely to result from PI-3K activity, which would have caused activation at an earlier time-point, as others have shown [167]. One confounder is that in differentiated Caco-2 cells, which are derived from colonic carcinoma, PI-3K activity may be constitutive. For example, there is evidence that PI-3K activity is increased in colonic and ovarian tumors [204-206]. 87 In view of these studies, it may be difficult to study the activation of PI-3K in Caco-2 cells, where high baseline activity (as seen in control untreated samples) could be masking the effects of F l iC-EAEC. Caco-2 cells therefore, may not be suitable as a model for studying PI-3K activation, at least by in-vitro kinase assay. Studies of PI-3K are necessary in another cell line, such as monocytic cell line THP-1, where its activation can be measured accurately. It is known that in THP-1 cells, Gram-negative flagellin activates TLR5 [65, 163] and involves IRAK [150]. However, flagellin induced signaling pathways distal to I R A K are not well defined in this cell line. When THP-1 cells were exposed to F l iC-EAEC, PI-3K activation was not detected by invitro kinase assay; however, Akt activation was noted in Western blots (Figure 19). Our data indicates that IL-8 secretion from Caco-2 cells incubated with FliC-E A E C involves the degradation of I-KB suggesting its regulation by another pathway besides PI-3K. A possible candidate for this is IRAK, which is an important enzyme in IL-1 and T L R signaling. Such a mechanism was shown in 3T3 fibroblast cell line, in which IL-1 induced activation of Akt was dependent on IRAK activation [207]. Based on this model, it may be proposed that Akt and N F - K B activation in Caco-2 cells incubated with F l iC-EAEC is regulated by IRAK. In support of this possibility, we noted that both Akt and N F - K B activation was seen at 20-30 min incubation with F l iC-EAEC. 4.2 Activation of p38 MAP kinase by FliC-EAEC in Caco-2 cells. Thus far, we have not identified a specific pathway that regulates F l iC-EAEC induced IL-8 secretion in Caco-2 cells. However, M A P kinase signaling has been shown to play an important role in cytokine secretion induced by microbial products in different cell lines. For example, the production of Th2 cytokines IL-5, IL-10 and IL-13 upon LPS 88 treatment of murine mast cells derived from bone marrow is mediated by the M A P kinases, c-Jun and p38. These effects were not observed in TLR4-deficient mice [208]. The interaction of lipoteichoic acid and CpG-DNA with TLR2 and TLR9 respectively, causes activation of N F - K B and M A P kinases in immune cells resulting in production of TNFa and IL-12 from macrophage cell lines and human dendritic cells [209]. In addition, Treponema lipoteichoic acid activation of M A P kinases p42/44 in myelo-monocytic cells is mediated by TLR4 [210]. These studies support a role for M A P kinases in TLR signaling. Although M A P kinases are generally regulated by M A P K K , another mechanism by which p38 M A P kinase is activated was found to be independent of M A P K K s . Instead, it involves TAB 1 (transforming growth factor-beta-activated protein kinase 1-binding protein 1), leading to formation of TRAF6-TABl-p38a complex and subsequent auto-phosphorylation of p38 [211]. Taken together, these data indicate that p38 M A P kinase activation by microbial products involves TLR signaling pathway. In view of this, we decided to undertake studies aimed at elucidating the role of p38 M A P kinase in IL-8 secretion by Caco-2 cells exposed to F l iC-EAEC. Lack of inhibition of IL-8 with Wortmannin raised the possibility that another signaling pathway is regulating Fl iC-EAEC induced IL-8 secretion. M A P kinases such as p38 M A P kinase and E R K 1 and 2 have been implicated in stimulus induced IL-8 secretion in epithelial cells [115, 212, 213]. Initially, we investigated the role of p38 M A P kinase in IL-8 secretion from Caco-2 cells exposed to F l iC-EAEC, by using two independent experimental approaches. Our first objective was to demonstrate that p38 M A P kinase is involved in IL-8 secretion due to F l iC-EAEC. 89 Lower concentrations of SB 203580 (3-10 p.M) were used to inhibit p38 M A P kinase activity, to decrease the possibility of affecting E R K . A review of the literature showed that the inhibitory dose of SB 203580 is variable, depending on the type of cell line and the end point under study. In THP-1 cells, pretreatment with 10 u M of SB 203580 completely blocked Clostridiuim difficile Toxin A induced IL-8 secretion [213]. In contrast, 5-10 uM did not influence Caco-2 migration in a study of intestinal wound healing and repair [214]. Similarly, SB 203580 did not affect phorbol ester-induced intestinal glutamine transport in the range of 0-10 uM in Caco-2 cells [215]. The above findings indicate that SB 203580 acts specifically on p38 M A P kinase in the range of 0-10 uM without affecting other cellular functions. In addition, we confirmed that SB 203580 was not toxic to Caco-2 cells by trypan blue staining as mentioned earlier. Pre-incubation of Caco-2 cells with lOuM SB 203580 abolished the secretion of IL-8 due to F l iC-EAEC (Figure 11), and a lower dose of 3uM produced a 50% decrease in IL-8 secretion due to two different doses of F l iC-EAEC (Figure 12). It is known that activation of p38 M A P kinase is preceded by phosphorylation at tyrosine and threonine residues [92]. Therefore, immunoprecipitation and Western analysis (Figures 13 and 14) provided further evidence of p38 M A P kinase activation in Caco-2. In Caco-2 cells, IL-1(3 also stimulated p38 M A P kinase and was included as a control. Maximal activation of p38 M A P kinase was noted at 30 minutes of F l iC-EAEC stimulation of Caco-2 cells (Figure 14). This time point is consistent with data from other studies [102, 219]. SB 203580 inhibition of p38 M A P kinase phosphorylation may explain its inhibition of Fl iC-E A E C induced IL-8 release (Figure 15). IL-l-induced phosphorylation of p38 M A P kinase was also blocked in Caco-2 cells by SB 203580. 90 The data presented in Figures 11-15 suggest that p38 M A P kinase activation is crucial for F l iC-EAEC induced IL-8 release from Caco-2 cells. It is tempting to speculate that activation of both p38 M A P kinase and N F - K B are under the regulation of a single mediator that functions upstream. Others have also described TLR mediated activation of N F - K B and M A P kinase. For example, bacterial fimbrae activate p38 M A P kinase and N F - K B , through a mechanism that utilizes TLR2 [220]. Interestingly, SB 203580 was shown to attenuate N F - K B dependent transcription due to IL-1 and LPS [221, 222], which suggests that p38 M A P kinase may directly regulate N F - K B . IRAK has been shown to regulate stimulus-induced activation of both N F - K B and p38 M A P kinase activation in different systems. For example, in mice lacking I R A K - M (a negative regulator of IRAK), LPS and CpG D N A caused excessive N F - K B and p38 M A P kinase activation [223]. Thus, F l iC-EAEC activation of TLR5 could lead to IRAK-mediated regulation of N F - K B and p38 M A P kinase. Elements of this pathway were shown to be involved in IL-8 secretion due to EHEC infection of Caco-2 cells [224]. Further, EHEC infection and exposure of Caco-2 cells to mutants lacking virulence factors led to N F - K B and p38 M A P kinase activation. Similarly, EHEC infection of T84 intestinal epithelial cells caused N F - K B and M A P kinase mediated IL-8 secretion [225]. Taken together, these data support a role for IRAK-mediated p38 M A P kinase and N F -KB activation due to F l iC-EAEC. Activation of p38 M A P kinase by Fl iC-EAEC is an example of a paradigm which suggests that the human intestinal epithelium has evolved to recognize bacterial surface structures which initiate a signal culminating in an inflammatory response in the host. 91 Therefore, an interesting question would be: Do cells of the immune system respond to Fl iC-EAEC by activating the p38 M A P kinase-signaling pathway? 4.3 Activation of Akt andp38 MAP kinase by FliC-EAEC in THP-1 cells. The data in Figures 16 and 17 indicate that incubation of THP-1 cells with FliC-E A E C leads to IL-8 secretion in a time- and dose-dependent manner. Pharmacological inhibition of p38 M A P kinase abrogated FliC-EAEC-induced IL-8 secretion from THP-1 cells (Figure 18). LPS induced IL-8 secretion was also susceptible to p38 M A P kinase inhibition. These findings are consistent with our earlier data that p38 M A P kinase regulates IL-8 secretion due to Fl iC-EAEC, whereas PI-3K activity is not a requirement. Others have shown that Vitamin D3-induced PI-3K activation can be measured in THP-1 cells by in-vitro kinase assay [187]. Since we were able to show PI-3K activation by LPS, it is unlikely that lack of PI-3K activity in THP-1 cells after F l iC-EAEC exposure, could be attributed to our technique. Instead, it is probable that F l iC-EAEC does not activate PI-3K in Caco-2 and THP-1 cells (Figure 19). Consistent with our earlier data, F l iC-EAEC activated Akt in THP-1 cells (Figure 19). However, it is not clear why this does not correlate with PI-3K activation in Caco-2 and THP-1 cells. It may be hypothesized that, perhaps, Akt is regulated by a mechanism that does not involve PI-3K activity, and instead, an unknown upstream mediator stimulates Akt upon Fl iC-EAEC exposure. However, such a mechanism for regulation of Akt has not been reported previously. We were also able to demonstrate the activation of p38 M A P kinase by Fl iC-EAEC in THP-1 cells (Figure 20), which is in agreement with our data from Caco-2 cells. 92 A review of the literature shows that while some investigators have used differentiated THP-1 cells, others have used the undifferentiated form to study host-pathogen interactions. A major difference between the two phenotypes is the expression of CD 14 co-receptor. Agents that induce differentiation of THP-1 cells such as P M A and Vitamin D3 increase the expression of CD 14 [187]. We found that these agents also induce significant IL-8 secretion from THP-1 cells. Therefore, we decided that undifferentiated THP-1 cells would be a better system in which to measure IL-8 secretion and to study F l iC-EAEC signaling. CD 14 is a required for signaling in THP-1 cells exposed to some bacterial products. For example, IL-1 secretion from THP-1 cells stimulated by LPS and L A M is dependent on CD 14 expression [226, 227]. Further, activation of N F - K B and p38 M A P kinase in THP-1 cells exposed to Staphylococcus aureus and Helicobacter pylori respectively, is dependent on CD14 expression [228, 229]. In contrast, it is not known if flagellin signaling also requires CD 14 expression. Our data generated in undifferentiated THP-1 cells, which express low levels of CD14 [187]is thus an interesting finding. The next objective was to determine whether there is a relationship between p38 M A P kinase-mediated IL-8 secretion and TLR signaling. In section 3.4, F l iC-EAEC signaling is examined in HEp-2-human epithelial cells transiently expressing TLR5. These cells were transfected with wild-type TLR5, and then incubated with F l iC-EAEC to assess the impact on IL-8 secretion and p38 M A P kinase activity. 4.4 Activation of p38 MAP kinase and IRAK in TLR5 transfected HEp-2 cells. The experiments in HEp-2 cells were designed to investigate the effect of Fl iC-E A E C treatment on TLR5 signaling pathways. TLR5 transfection of HEp-2 cells 93 increased the magnitude of IL-8 secretion in response to F l iC-EAEC (Figure 21). Increased IL-8 secretion required p38 M A P kinase activation, as inhibition of p38 M A P kinase abrogated secretion of IL-8 from TLR5-transfected cells (Figure 22). Importantly, activation of p38 M A P kinase was seen in Western analysis of TLR5-transfected HEp-2 cells exposed to F l iC-EAEC (Figure 23). Together, these data suggest that in TLR5-transfected cells, activation of p38 M A P kinase is required for IL-8 secretion following exposure to F l iC-EAEC. Several TLRs contain Y X X M motifs in their cytoplasmic domain that are putative sites for tyrosine phosphorylation and binding of PI-3K via its SH2 domain during activation of TLRs [230]. A Y X X M motif that binds PI-3K has been described for TLR1, TLR2 and TLR6 [228]. IL-1R1 and MyD88 also contain such motifs that are possibly important for signaling due to TLR activation. The cytoplasmic domain of TLR5 contains an Y X X M motif at position 798; the role of this tyrosine residue in signaling is unknown. A mutant TLR5 in which a leucine was substituted for tyrosine at position 798 (Y798L) was generated in our laboratory to determine whether tyrosine phosphorylation of this motif in TLR5 was required for F l iC-EAEC signaling. Secretion of IL-8 was abolished in HEp-2 cells transfected with the Y798L TLR5 (Figure 24), suggesting that tyrosine phosphorylation of TLR5 is necessary for IL-8 secretion from HEp-2 cells. Since activation of p38 M A P kinase was still detectable in mutant TLR5 due to F l iC-EAEC (Figure 25), it appears that p38 M A P kinase activation in TLR5 transfected cells is independent of this tyrosine motif in the receptor. It is not known if tyrosine phosphorylation in TLR5 is necessary for its activation or binding to other signaling proteins, as described for TLR4. LPS induction of IL-8 94 secretion from P B M C requires tyrosine phosphorylation of TLR4 [231]. Although experiments with Y798L implicate PI-3K, activation of PI-3K by F l iC-EAEC was not detected in Caco-2 and THP-1 cells. Furthermore, inhibition of PI-3K did not influence IL-8 secretion from Caco-2 cells exposed to Fl iC-EAEC. It is therefore unlikely that phosphorylation of tyrosine (798) in TLR5 leads to activation of PI-3K. Alternatively, it may be speculated that phosphorylated tyrosine (798) binds another adapter molecule in the TLR signaling pathway such as MyD88, leading to IL-8 secretion from Caco-2 cells incubated with F l iC-EAEC. We have shown above that TLR5 transfection of HEp-2 cells makes them competent to respond to F l iC-EAEC by activation of p38 M A P kinase leading to IL-8 secretion. Since IRAK is an important mediator of TLR signaling, we examined the activation of IRAK in Caco-2 and TLR-5-transfected Hep-2 cells exposed to Fl iC-EAEC. An antibody that binds the phosphorylated species of IRAK was used in Western blots. Others have shown that TLR agonist-induced IRAK activation involves phosphorylation at serine/threonine residues, recruitment to the TLR and eventual degradation by ubiquitination [80, 232]. We found that F l iC-EAEC treatment of Caco-2 cells led to IRAK activation, followed by its degradation (Figure 26). Activation of IRAK was also seen due to IL-1 treatment in Caco-2 cells. Experiments in HEp-2 cells showed increased IRAK activity in TLR5-transfected cells exposed to F l iC-EAEC (Figure 27). Although TLR5 transfection of HEp-2 cells confers IL-8 secretion in response to Fl iC-EAEC [37], we have not determined whether transfected HEp-2 cells express TLR5 mRNA or protein. Furthermore, we have not shown that transfected cells express TLR5 on their cell membranes. In the absence of these data, our transfection model has 95 limitations; however, studies are being undertaken in our laboratory with a view to detect TLR5 mRNA and protein in transfected HEp-2 cells and to establish the role of TLR5 in F l iC-EAEC signaling. In summary, we have demonstrated that TLR5 transfection of HEp-2 cells leads to IL-8 secretion by Fl iC-EAEC. This is mediated by p38 M A P kinase and IRAK activation in Caco-2 and TLR5-transfected HEp-2 cells. Tyrosine phosphorylation of TLR5 may be required for F l iC-EAEC induced IL-8 release in transfected HEp-2 cells. These data support our hypothesis that TLR5 mediates FliC-EAEC-induced signaling. 9 6 Chapter V: Conclusions and future directions 5.1 PI-SK/Akt signaling by FliC-EAEC The involvement of PI-3K-mediated signaling pathways induced by F l iC-EAEC was investigated in Caco-2 intestinal epithelial and THP-1 monocytic cells. The results revealed a two-fold increase in PI-3K activity, and a ten-fold activation of its target Akt, in Caco-2 cells following Fl iC-EAEC exposure. In spite of this, pharmacological inhibitors of signaling enzymes indicated that PI-3K was not required for IL-8 release from Caco-2 and THP-1 cells. Similarly, experiments with U937 cells containing a mutant p85 subunit of PI-3K showed that this lipid kinase is not necessary for chemokine secretion. The biological consequences of F l iC-EAEC induced PI-3K/Akt activation may include transcriptional activation of IL-2, differentiation and/or survival of intestinal epithelial cells [203]. For example, phospho-activation of Akt due to IL-1 [3 has been demonstrated in fibroblasts and thymoma cell lines [207]. Further, it has been shown that inhibition of PI-3K enhances enterocyte-like differentiation of Caco-2 cells [176]. In this model, activation of Akt by Fl iC-EAEC may delay the differentiation of Caco-2 cells into enterocytes, therefore making the epithelial cells prone to E A E C infection. Another study suggested that PI-3K activation negatively regulates TRAIL (TNF-related apoptosis-inducing ligand) receptor expression in Caco-2 cells [176]. It may be hypothesized that F l iC-EAEC activation of Akt might influence the survival of Caco-2 cells by decreasing expression of TRAIL receptors. Secretion of IL-2 is known to be restricted to cell lines derived from T cells, and IL-2 along with IFNy and TNFa are considered to be Thl cytokines [233]. Interestingly, 97 Rodriguez-Juan et al have recently shown that IL- lp increases RANTES production and IL-2 transcription in Caco-2 cells, although IL-2 protein was unaffected [234]. The authors of this study mentioned that instability of IL-2 mRNA could possibly account for the lack of increased IL-2 secretion. As mentioned earlier, p38 M A P kinase activity influences the stability of IL-8 mRNA following stimulation with inflammatory agonists [92]. Rodriguez-Juan et al did not examine p38 M A P kinase, and it is not known whether its activation would have enabled Caco-2 cells to secrete IL-2. We have seen activation of Akt and p38 M A P kinase by Fl iC-EAEC in Caco-2 cells. It is tempting to hypothesize that activation of Akt and p38 M A P kinase may regulate IL-2 secretion from Caco-2 cells. The secretion of IL-2 (a Thl cytokine) may lead to development of Thl immune response which is likely to eradicate infections in the host, whereas Th2 responses are associated with persistence of infectious agents [144]. Data from Caco-2 cells suggested that transcription factor N F - K B is involved in the molecular mechanism that regulates F l iC-EAEC induced IL-8 release. The activation of N F - K B may be due to TLR signaling pathway. A link between PI-3K and IRAK was discovered during IL-1 induced IL-2 secretion from a thymoma cell line. In these cells, PI-3K/Akt-mediated IL-2 release was shown to be dependent on I R A K [207]. It is possible that N F - K B mediated IL-8 secretion due to F l iC-EAEC may be regulated by IRAK in Caco-2 cells. In this model, IRAK activation may lead to TRAF6 mediated 1-KB degradation and N F - K B nuclear migration. A similar mechanism has been postulated for heat shock protein-HSP70, which caused inflammatory cytokine production mediated by MyD88/IRAK and N F - K B [146] and in other studies . 98 In summary, we have shown that N F - K B activation contributes to IL-8 secretion from Caco-2 cells incubated with F l iC-EAEC. IL-8 secretion did not require PI-3K, since we were unable to show its activation by in-vitro kinase assay. Furthermore, use of a dominant negative mutant of PI-3K or inhibition of endogenous PI-3K by Wortmannin did not decrease FliC-EAEC-induced IL-8 secretion. However, we found that F l iC-EAEC activates Akt in Caco-2 and THP-1 cells. FliC-EAEC-induced activation of Akt is not necessary for IL-8 secretion and its biological significance, therefore, remains unknown at present. 5.2 Regulation of FliC-EAEC-induced IL-8 secretion by p38 MAP kinase activation The role of p38 M A P kinase in FliC-EAEC-induced IL-8 secretion was investigated by three independent experimental methods. Pharmacological inhibition of p38 M A P kinase abolished the release of IL-8 in a dose- and time-dependent manner from Caco-2, THP-1 and TLR5-transfected Hep-2 cells. Results from these experiments suggest that p38 M A P kinase regulates IL-8 secretion by undergoing phospho-activation. Our data is consistent with the findings in a recently study in which flagellin from S. typhimurium activated TLR5 mediated activation of p38 M A P kinase leading to IL-8 secretion from intestinal epithelial cells [164]. Activation of p38 M A P kinase is critical for propagation of pro-inflammatory signals initiated by Fl iC-EAEC. Pharmacological agents that modify p38 M A P kinase activity may alter the development of the innate immune response in some diseases where excessive or diminished TLR signaling is a causative factor. The clinical relevance of M A P kinase signaling is that they are an attractive target for drug development in treatment of chronic inflammatory disease such as rheumatoid arthritis and inflammatory 99 bowel disease [235]. A potential anti-inflammatory compound is CNI-1493, a synthetic guanylhydrazone that inhibits the phosphorylation of both p38 M A P kinase and INK [236]. CNI-1493 can suppress macrophage activation and production of pro-inflammatory cytokines including TNFa, IL-1 and IL-6 [237, 238]. A large multi-centre study is currently underway to evaluate the clinical usefulness of this drug. p38 M A P kinase is therefore unique because it is the only kinase, which is currently targeted by a drug for treatment of inflammatory conditions. 5.3 FliC-EAEC activates TLR5 signaling pathways in epithelial cells Although TLR5 is known to confer IL-8 secretion from epithelial cells exposed to Fl iC-EAEC [37], the signaling mechanisms resulting from its activation remain unknown. Experiments in HEp-2 cells transiently expressing TLR5 were performed to dissect the signaling pathways activated upon exposure to F l iC-EAEC. Transfection of HEp-2 cells with TLR5 led to IL-8 secretion after exposure to F l iC-EAEC. Secretion of IL-8 was abolished by pharmacological inhibition of p38 M A P kinase. Further, p38 M A P kinase activation was observed in TLR5-transfected HEp-2 cells incubated with Fl iC-EAEC. To our knowledge, these results are first evidence of flagellin from E A E C inducing activation of p38 M A P kinase via TLR5 in epithelial cells. A n increase in IL-8 secretion following TLR5-transfection was not seen when HEp-2 cells were transfected with a mutant (Y798L) TLR5 and exposed to Fl iC-EAEC. However, p38 M A P kinase activation was still noted in these cells. These findings suggest that activation of p38 M A P kinase through TLR5, in response to F l iC-EAEC is independent of Y798L. However, tyrosine phosphorylation of Y798L is required for IL-8 100 secretion from HEp-2 cells exposed to F l iC-EAEC. This implies involvement of other signaling enzymes. One such signaling enzyme is IRAK. Since IRAK activation is pivotal in TLR signaling [239], we performed studies in Caco-2 and TLR5-transfected HEp-2 cells to determine whether IRAK was activated by F l iC-EAEC. We found that incubation of F l iC-EAEC with Caco-2 and TLR5-transfected cells caused phospho-activation of IRAK, as measured by Western blots. This suggests the possibility that activation of IRAK by Fl iC-EAEC is important for distal signaling events such as p38 M A P kinase activation. Based on the above data, we have shown that F l iC-EAEC signaling involves TLR5 signaling mediated by p38 M A P kinase dependent IL-8 secretion. Activation of IRAK is further evidence of involvement of TLR5 in this signaling pathway. 5.4 Implications of FliC-EAEC signaling in EAEC diarrhea To summarize, results reported in this thesis show that IL-8 secretion from intestinal epithelial and monocytic cells exposed to F l iC-EAEC is dependent on p38 M A P kinase activation. This is likely due to the interaction of F l iC-EAEC with TLR5 and IRAK activation. The activation of these signaling enzymes reflects important events in host immune response during E A E C diarrhea. These findings highlight the critical role of TLRs in inflammation and diarrhea that characterize E A E C infection. Importantly, what we have learned is that p38 M A P kinases are key regulators of inflammation, and that activation of p38 is a pre-requisite for the commencement of immune response in host. However, we do not know if these signaling mechanisms are functional in-vivo. Since IL-8 is a chemokine for neutrophils, evidence of neutrophilic infiltration has been found in stool samples of some patients with E A E C diarrhea [22, 132]. Therefore, 101 our studies describing mechanisms of IL-8 secretion due to F l iC-EAEC in epithelial cells, complement other studies that have found markers of inflammation in E A E C diarrhea. Neutrophils may act by disrupting the intestinal epithelial barrier and contribute to fluid secretion by releasing cAMP. FliC-EAEC-induced IL-8 secretion is likely to cause inflammation in a similar manner during E A E C diarrhea. In addition to the role of TLR signaling in inflammation, these receptors also play an important role in the development of T helper cells (Thl), so that the immune system is programmed to eradicate pathogens during infection. One may speculate that Fl iC-E A E C induced signaling pathways mediated by p38 M A P kinase are involved in this process, especially in children with E A E C diarrhea. One possible outcome of p38 M A P kinase activation, in the long term, may lead to a decrease the likelihood of developing atopy and allergic diseases such as asthma (mediated by Th2). Interestingly, M A P kinases (including p38 M A P kinase), are attractive targets for therapy in the treatment of asthma and autoimmune diseases [240] and inhibitors of M A P kinases are being investigated as potential drugs to treat human diseases in future [241]. A proposed model of F l iC-EAEC signaling pathways, summarizing the important findings in this thesis is shown in Figure 28. 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