| Summary: | The integrity of the intestinal epithelium is maintained by intercellular junctions, such as tight junctions. These are crucial in creating a barrier against the intestinal environment. Studies have shown that pathogenic bacteria can disrupt the structure of tight junction proteins to facilitate their paracellular passage into the underlying tissues. It is known that colonisation of the intestinal epithelium by Campylobacter affects intestinal integrity by disrupting occludin, an integral tight junction protein, enhancing the paracellular passage or Campylobacter. In response to the influx of foreign antigens, the host will mount an immune response elevating the levels of proinflammatory cytokines, such as TNF-a and IFN-y. Such cytokines have been shown to affect the structure of tight junctions during intestinal inflammation disrupting barrier function and facilitating the passage of luminal antigens into the underlying tissues. Published data show that some bacteria are capable of elevating cytokine levels by manipulating the host immune response, such synergistic interactions enhanced the pathogenesis of infection observed. This study was undertaken to examine how Campylobacter jejuni interacts with host epithelial cells and to determine if synergistic interactions with the products of the host immune response and/or the microbial community enhanced the pathogenesis of infection observed. In vitro Caco-2 monolayers were pre-incubated with inhibitors of the host cytoskeleton one hour prior to C. jejuni infection. Throughout the time course transepithelial electrical resistance (TER) readings were recorded at 24 hour intervals and levels of transcytosed bacteria determined. Monolayers were fixed at 24 hour time intervals and stained for occludin and E-actin. Caco-2 mono layers were inoculated with combinations of C. jejuni 81116 and/or proinflammatory cytokines and/or E. coli isolates over a time course. Throughout the time course, TER readings were recorded at intervals and levels of translocated bacteria in the basal well determined. Monolayers were fixed and stained for occludin, cell viability was determined using a tenninal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay. Gnotobiotic chicks were infected in vivo with combinations of C. jejuni 81116 and/or E. coli isolate eight. Two days post infection, chicks were sacrificed and tissues harvested. Bacterial colonisation was determined by serial dilution onto selective agar. Tissue sections were stained with haematoxylin and eosin, internalised bacteria were determined using fluorescence in situ hybridisation. C. jejuni is capable of endocytosis into host eukaryotic cells but depending on the strain it has being shown to be a microtubule and/or micro filament dependent process. Thus Caco-2 cells were pre-exposed to inhibitors of the host cytoskeleton to determine which route C. jejuni 81116 utilised for endocytosis. Inhibition of either microfilaments and/or microtubules initially prevented endocytosis, but by 24 hours post infection levels of endocytsosed Campylobacter were equivalent to C. jejuni infection alone. Interestingly, when both microfilaments and microtubules were subsequently inhibited C. jejuni was endocytosed into the cells at a significantly higher level (P < 0.05) than seen with C. jejejuni infection alone, suggesting that endocytosis occurs independently of the host cytoskeleton. In response to Campylobacter infection the levels of proinflammatory cytokines would be enhanced which have being linked in previous studies to an increase in the permeability of the epithelium. Indeed, preliminary experiments conducted in this study hewed that C. jejuni 81116 in the presence of IFN-y alone or in the presence of TNF-a resulted in focal redistribution of occludin into an intracellular location and an increase in cellular damage within 24 hours, which was not seen with C. jejuni infection alone, indicating synergistic interactions. Subsequent experiments performed using inhibitors of the host cytoskeleton tentatively suggests a role for actin in the reduced association of occludin with tight junction complexes. Synergistic interactions were also indicated in vitro and in vivo when cells were exposed to C. jejuni and E. coli isolates. Indeed eo-infection in vitro caused a significant decrease in TER within 6 hours, with a focal redistribution of occludin into an intracellular location correlating with an influx of C. jejuni into the basal well. When the experiment was conducted in gnotobiotic chicks although no outward signs of disease were observed microbiological testing confirmed that both C. jejuni and E. coli could be found throughout the gastrointestinal tract, with extensive damage to all sites of the gastrointestinal tract sampled. These results suggest that the endocytosis of C. jejuni into host epithelial cells can occur independently of the host cytoskeleton. Once Campylobacter has colonised the iastrointestinal tract of a susceptible host infection the resulting intestinal inflammation in conjunction with C. jejuni infection would result in rapid loss of tight junction barrier function. Furthermore the polymicrobial environment of the gastrointestinal tract may further enhance the pathogenesis of C. jejuni infection observed. Both synergistic interactions with products of the host immune response and microbial community may play a role in the development of bloody diarrhoea and Campylobacter-mediated colitis.
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