Campylobacter jejuniCytolethal Distending Toxin Mediates Release of Interleukin-8 from Intestinal Epithelial Cells

Abstract

Campylobacter spp. are among the most common causes of human bacterial diarrhea worldwide. While campylobacter infections are quite common and often severe, relatively little is known about mechanisms of pathogenesis. Campylobacters are generally considered invasive, and invasiveness appears to be associated with disease in the ferret diarrheal disease model (3, 42). In addition, numerous cytotoxins in campylobacters have been described (39), but only cytolethal distending toxin (CDT) has been well characterized (29,30,40). CDT has been found in a variety of other bacteria including Escherichia coli (17, 33), Shigella spp. (26), Haemophilus ducreyi (12), Actinobacillus actinomycetemcomitans (25,35,36), and Helicobacter hepaticus (43). Although CDT has been shown to block eukaryotic cells in the G 2 phase of the cell cycle (6,28,40,43), its role in disease caused by such a diverse group of pathogens remains unclear. However, there is some evidence suggesting a role for CDT in diarrheal disease. An epidemiological study in Bangladesh showed a trend towards increased numbers of CDTpositive E. coli cells in diarrheal cases compared to asymptomatic controls, but the difference did not reach statistical significance (1). When fed to suckling mice, partially purified CDT from Shigella dysenteriae produced watery diarrhea and tissue damage in the descending colon (26).Campylobacter entercolitis is typically associated with a local acute inflammatory response and involves intestinal tissue damage. It is thought that the host inflammatory response may mediate many of the clinical symptoms (20), and inflammatory cytokine responses are recognized components of enteric infections. Interleukin-8 (IL-8) is a proinflammatory cytokine, a potent chemotactic factor for many immune effector cells, and a mediator of localized inflammatory responses. Helicobacter pylori, a primary cause of gastritis in humans, is known to induce IL-8 release from epithelial cells (15,34). Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Shigella spp. have also been shown to elicit IL-8 secretion from intestinal epithelial cells in vitro during invasion (9, 19). We have previously shown that Campylobacter jejuni also induces IL-8 secretion from intestinal epithelial cells by a process which correlated with adherence and/or bacterial invasion (14). In this report we demonstrate that C. jejuni mediates IL-8 secretion from intestinal epithelial cells by multiple mechanisms. One of these mechanisms, as previously described, involves adherence and/or invasion (14), while a second mechanism is mediated directly by CDT. MATERIALS AND METHODSBacterial strains and growth conditions. Campylobacters were routinely grown on Mueller-Hinton (MH) agar (Difco, Detroit, Mich.) under microaerobic conditions or in biphasic MH cultures. MH medium was supplemented with kanamycin to a final concentration of 50 g/ml or chloramphenicol at a final concentration of 20 g/ml in some cases. For counterselection of E. coli DH5␣ (RK212.1) donors (11, 21) in comp...

Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells RequiresCampylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-κB

“…TNF-␣ secreted by host epithelial cells can stimulate IL-8 secretion by intestinal epithelial cells (12). However, we showed that a TNF-␣ neutralization antibody that effectively (22). Our finding that the conditioned supernatant of the C. jejuni 81-176 cdtB mutant exhibited a ϳ50% reduction in IL-8 induction further confirms this notion and also suggests that additional factors are required for IL-8 induction.…”

“…However, the flagellin of C. jejuni has been reported to be ineffective in the induction of IL-8 secretion in human intestinal epithelial cells (27,58). Campylobacter-secreted cytolethal distending toxin (CDT) can induce IL-8 secretion (22). Additionally, C. jejuni-induced IL-8 secretion requires the viability of the bacteria (9, 21) and depends on C. jejuni gene products that are expressed upon contact with epithelial cells and on the activation of the mitogen-activated protein kinase extracellular signal-regulated kinase in epithelial cells (58).…”

Lack of response of INT-407 cells to the presence of non-culturableCampylobacter jejuni

“…The more adhesive and invasive strains, appeared to be the ones which also induced the highest levels of IL-8 in INT-407 cells, as previously shown by Hickey et al [28,29]. Next to strain-dependency, the level of adhesion and invasion was also found to be dose dependent as previously shown [13][14][15].…”

“…As the IL-8 assay is based on measuring an immune response, even if non-culturable C. jejuni do not recover, their effect on the infectivity is likely to be measured by the IL-8 assay. As in vitro studies have established that the invasive and adhesive ability of C. jejuni strains differ [21][22][23][24][25][26][27] and, furthermore, that the C. jejuni-stimulated IL-8 secretion is strain dependent [28,29] four C. jejuni strains were selected. C. jejuni 70.2 and BF were chosen as for these strains the formation of infective non-culturable C. jejuni has been described by Cappelier et al [9,30].…”

Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

“…The recent emergence of antibiotic-resistant strains has triggered renewed interest for the understanding of the pathogenesis of C. jejuni (2,3), which could lead to the identification of novel targets that could be further investigated for the development of therapeutics. A variety of virulence factors has been identified, such as the lipo-oligosaccharide (4 -7), capsule (8), flagellum (9), toxins (10), and adhesins (11)(12)(13)(14). They allow for colonization and invasion of the intestinal epithelium and protection against host immune defense systems.…”