Vibrio choleraeFlagellins Induce Toll-Like Receptor 5-Mediated Interleukin-8 Production through Mitogen-Activated Protein Kinase and NF-κB Activation

Abstract

The gram-negative bacterium Vibrio cholerae causes cholera, an acute diarrheal disease characterized by voluminous rice water stools and rapid dehydration. V. cholerae produces an ADP-ribosylating enterotoxin called cholera toxin (CT), encoded by ctxA and ctxB, that activates the host cell enzyme adenylate cyclase, resulting in profuse diarrhea in humans (15). The preeminence of CT as a major virulence factor of cholera was confirmed in volunteer studies, making this toxin a prime target in the development of cholera vaccine strains (21,23). While the first generation of ⌬ctxA and ⌬ctxAB V. cholerae vaccine strains did not cause cholera, they nevertheless caused significant reactogenicity in individuals in the form of mild to moderate diarrhea (23). Although the reason for the reactogenicity of the ⌬ctx vaccine strains is unknown, two hypotheses were introduced as possible explanations (22). First, V. cholerae may produce additional unidentified enterotoxins that were previously undetectable because of the dominant effects of CT. Second, adherence of V. cholerae during colonization of the proximal small intestine may cause alterations in small intestine function, resulting in diarrhea. Although further efforts to improve live oral cholera vaccines have focused on deleting newly characterized toxins, the reactogenicity in these strains was not markedly diminished (9, 49, 52). This observation supports the latter hypothesis.While cholera is not generally considered an inflammatory disease, there is evidence of inflammation in cholera patients and from human and animal vaccine studies. For instance, immune cell infiltration and activation have been observed in patients with cholera (10,24,33,34). More recently, Qadri et al. reported neutrophil infiltration into the lamina propria along with an increase in inflammatory mediators, such as tumor necrosis factor alpha, in adults and children during the acute stage of V. cholerae O1 and O139 serogroup infections (35,36). In human volunteer studies, the fecal lactoferrin levels induced by the reactogenic ⌬ctx vaccine strain CVD110 were markedly higher than those induced by the wild-type CT-expressing El Tor strain (44). The lactoferrin levels induced by CVD110 were comparable to elevated levels found in volunteers who ingested Shigella, the prototypic inflammatory enteric pathogen (30). Lactoferrin is a major component of polymorphonuclear leukocytes and is secreted by most mucosal membranes during inflammatory responses. Its presence in feces is indicative of leukocyte infiltration and intestinal inflammation. In a rabbit model, elevated levels of interleukin-1␤ (IL-1␤) and IL-8 were observed following infection with two different reactogenic ⌬ctx V. cholerae vaccine strains (E. C. Boedecker and J. B. Kaper, unpublished observations). IL-8 production has also been reported to contribute to the recruitment of neutrophils following infection with the enteric pathogens Salmonella spp. and Escherichia coli (4, 25, 42). Reacto-* Corresponding author. Mailing address:

Reactogenicity of live-attenuated Vibrio cholerae vaccines is dependent on flagellins

“…Flagellins have been found to interact with TLR5 and to trigger MyD88-and NF-κB-dependent transcription of proinflammatory cytokines (19). Notably, all five V. cholerae flagellins, which can be secreted independently of flagellum filament assembly, contain the amino acid motif that stimulates TLR5, and purified V. cholerae flagellins were found to elicit TLR5-dependent IL-8 secretion from T84 cells (10). In addition, flagellins have been shown to activate the NLRC4 inflammasome, promoting IL-1β maturation and secretion (20)(21)(22).…”

“…The effect of these factors might be potentiated by close contact between the bacteria and the epithelium. Finally, recent work using tissue-culture models has led to the hypothesis that the five V. cholerae flagellins, which have been demonstrated to activate the Toll-like receptor 5 (TLR5) signaling pathway, could lead directly to reactogenic diarrhea by stimulating production of proinflammatory cytokines in the intestine (10,11). Detection of lactoferrin and fecal leukocytes in the stools of volunteers with reactogenic diarrhea (12,13) supports the idea that intestinal inflammation is associated with vaccine reactogenicity.…”

“…Bacterial flagellin proteins, including all five V. cholerae flagellins, are known to stimulate production of proinflammatory cytokines by activation of TLR5 (10,11,18). We therefore compared the relative abundance of transcripts for several cytokines in tissue homogenates from rabbits infected with Peru-NT or Peru-NTΔflaABCDE and from mock-infected rabbits.…”

Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

“…These studies correlate with clinical observations in which naturally occurring "nontoxigenic" V. cholerae O1 strains that have not acquired the CTX⌽ prophage induce diarrhea that is more inflammatory (5,33). In an infant rabbit model of infection, this diarrhea has been specifically linked to expression of flagellin that may be functioning by increasing inflammatory signaling through TLR5 (20,46,47). Studies using lung infection in mice have also linked proinflammatory signaling from CT-negative strains to LPS-dependent signaling through TLR4 (18), whereas lipoprotein has been shown in vitro to stimulate inflammation via TLR1/TLR2 (16,25).…”

Interleukin‐8 gene regulation in epithelial cells by Vibrio cholerae: role of multiple promoter elements, adherence and motility of bacteria and host MAPKs

“…We reported that motility and adherence to intestinal epithelial cells were the triggering factors contributing to IL-8 expression by V. cholerae [13]. Vibrio cholerae flagellins and purified flagella were found to induce IL-8 secretion in vitro [15,16]. Vibrio cholerae flagellins, regardless of whether the proteins were assembled into a flagellum or the flagellum was functional could also elicit IL-8 in vivo [17].…”