Gangliosides act as co-receptors for Salmonella enteritidis FliC and promote FliC induction of human beta-defensin-2 expression in Caco-2 cells

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-kappaB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521-30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.     

Production of beta-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein.

We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis toxin. The FliC-induced increase in hBD-2 promoter activity via nuclear factor kappaB (NF-kappaB) was also inhibited by chelation of intracellular Ca(2+) or by U73122. We conclude that FliC increased [Ca(2+)](in) via inositol 1,4,5-trisphosphate, which was followed by up-regulating hBD-2 mRNA expression via an NF-kappaB-dependent pathway.     

Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.

Helicobacter pylori is an etiological agent of gastritis, peptic ulcer, and gastric cancer. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which belongs to one of the most important host defense systems against bacterial infection in several epithelial tissues. We studied the effect of H. pylori on the expression of hBD-2 mRNA in MKN45 gastric mucosal cells. H. pylori, but not culture filtrate, increased the hBD-2 mRNA level in MKN45 cells; the inductive effect of H. pylori was not detected with Intestine 407 cells. Among H. pylori strains, strain OHPC0002, which lacks a cag Pathogenicity Island (PAI), did not induce hBD-2 mRNA in MKN45 cells. These results suggested that H. pylori cag PAI is critical for the induction of hBD-2 mRNA in MKN45 cells. Exposure of MKN45 cells to Salmonella typhimurium, S. enteritidis, S. typhi, and S. dublin, but not Escherichia coli ML35, also resulted in induction of hBD-2 mRNA.     

Virulent strains of Salmonella enteritidis disrupt the epithelial barrier of Caco-2 and HEp-2 cells

To confirm the existence in nature of Salmonella enteritidis strains of different degrees of virulence and to elucidate the mechanisms underlying the effects of such strains on the epithelial barrier function, the consequences of infection of Caco-2 cells and HEp-2 cells with 15 S. enteritidis strains in a chicken infection model were examined. The more virulent strains of S. enteritidis, which are biofilm producers in adherence test medium, were able to disrupt HEp-2 and Caco-2 monolayers, as shown by transmonolayer electrical resistance and lactate dehydrogenase activity. In contrast, the low-virulence strains of S. enteritidis, which do not produce biofilms in adherence test medium, had no effect on the same cells. An avirulent rough mutant of Salmonella minnesota exhibited a pattern of behaviour similar to that of the low virulence strains of S. enteritidis, whilst a clinical Salmonella typhi strain caused rapid injury to the monolayers. The effect of supernatants of Salmonella cultures in adherence test medium on the integrity of Caco-2 cell monolayers indicated that the high-virulence S. enteritidis strains, but not the low-virulence strains, release a soluble factor when incubated under optimum biofilm-forming conditions, which enables the disruption of the integrity of Caco-2 monolayers.     

NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2

Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frame-shift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-kappaB inhibitor Helenalin as well as a super-repressor form of the NF-kappaB inhibitor IkappaB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated down-regulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.     

Interference ofSalmonella enteritidis andLactobacillus spp. with IL-8 levels and transepithelial electrical resistance of enterocyte-like caco-2 cells

Caco-2 cells (exhibiting characteristics of mature villus enterocytes) were used to determine bacteria (Salmonella enteritidis causing human gastroenteritis)-intestinal cell interactions. The interference of bacteria with the transepithelial electrical resistance (TEER) of filter-grown Caco-2 cells and the production of IL-8 after exposure of the cells to S. enteritidis 857 and/or Lactobacillus strains (L. gasseri LF221 and L. rhamnosus BGT10) was evaluated. The strain 857 decreased TEER of filter-grown Caco-2 cells; in contrast, lactobacilli had a little or no effect. The effect of S. enteritidis on the TEER decreased if Caco-2 cells were pre-incubated with lactobacilli. This strain induced high levels of IL-8 (which can lead to cell damage). Compared to the IL-8 synthesis after exposure of Caco-2 cells to S. enteritidis 857, simultaneous exposure of Caco-2 cells to S. enteritidis and lactobacilli inhibited the IL-8 synthesis after short recovery periods.     

Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S. enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S. enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40-43 degrees C) or to graded numbers (10(1)-10(8)) of bacteria and in villus-like cells exposed to S. enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S. enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S. enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.     

Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic

The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particular Staphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human beta-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureus revealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor alpha and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.     

Regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-kappaB transcription factor family.

Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the beta-defensin family. Previous work has shown that multiple signaling pathways are involved in human beta-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with a periodontal bacterium, Fusobacterium nucleatum, and other stimulants. The goal of this study was to further characterize these pathways. The role of NF-kappaB in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-kappaB activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-kappaB in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-kappaB inhibitors, pyrrolidine dithiocarbamate and the proteasome inhibitor, MG132. To investigate alternative modes of hBD-2 regulation, we explored involvement of mitogen-activated protein kinase pathways. F. nucleatum activated p38 and c-Jun NH(2)-terminal kinase (JNK) pathways, whereas it had little effect on p44/42. Furthermore, inhibition of p38 and JNK partially blocked hBD-2 mRNA induction by F. nucleatum, and the combination of two inhibitors completely blocked expression. Our results suggest that NF-kappaB is neither essential nor sufficient for hBD-2 induction, and that hBD-2 regulation by F. nucleatum is via p38 and JNK, while phorbol ester induces hBD-2 via the p44/42 extracellular signal-regulated kinase pathway. Studies of hBD-2 regulation provide insight into how its expression may be enhanced to control infection locally within the mucosa and thereby reduce microbial invasion into the underlying tissue.     

Toll-like receptor 2-mediated expression of beta-defensin-2 in human corneal epithelial cells

We previously showed that human corneal epithelial cells (HCECs) express Toll-like receptors (TLRs), which recognize gram-positive bacteria and respond to Staphylococcus aureus infection by the expression and secretion of proinflammatory cytokines and beta-defensin-2 (hBD2). In this study, we further elucidated the underlying mechanisms regulating hBD-2 expression and its role in innate defense in HCECs in response to S. aureus challenge. Exposure of HUCL cells, a telomerase-immortalized HCEC line, to S. aureus, its exoproducts (1:10 dilution), or synthetic lipopeptide Pam3Cys (10 microg/ml) resulted in the up-regulation of hBD-2, but not hBD1 and hBD3. Similar to HUCL cells, primary HCECs responded to S. aureus-exoproducts and Pam3Cys challenge by expressing hBD2 mRNA and secreting hBD2 into the culture media. Furthermore, these stimuli induced the expression of TLR2 at both mRNA and protein levels. Consistently with its role as a major pattern-recognizing receptor, TLR2 was located at the cell surface by cell surface biotinylation. The treatment of HUCL cells with TLR2 neutralizing antibody resulted in a significant decrease in Pam3Cys-induced hBD2 production as well as IL-6, IL-8, and TNF-alpha secretion. The Pam3Cys-induced hBD2 expression was completely blocked by NF-kappaB inhibitors and partially inhibited by p38 MAP kinase and the JNK inhibitors. Conditioned media derived from HCECs challenged with S. aureus-exoproducts or Pam3Cys exhibited antibacterial activity against S. aureus, Pseudomonas aeruginosa and Escherichia coli. These findings suggest that S. aureus induces hBD2 production through TLR2-mediated pathways in HCECs and that pathogen-challenged, TLR-activated HCECs possess antimicrobial activity. Thus, the epithelium might play a role in innate defense against bacterial infection by directly killing bacteria in the cornea.     

Requirements for two proximal NF-kappaB binding sites and IkappaB-zeta in IL-17A-induced human beta-defensin 2 expression by conducting airway epithelium

Among a panel of 21 cytokines (IL-1alpha, -1beta, -2-13, and -15-18; interferon-gamma; granulocyte-macrophage colony-stimulating factor; and tumor necrosis factor alpha), we have recently observed that IL-17A is the most potent inducer for human beta-defensin 2 (hBD-2) in conducting airway epithelial cells (Kao, C. Y., Chen, Y., Thai, P., Wachi, S., Huang, F., Kim, C., Harper, R. W., and Wu, R. (2004) J. Immunol. 173, 3482-3491). The molecular basis of this regulation is not known. In this study, we demonstrated a coordinated degradation of inhibitory kappaB(IkappaB)-alpha followed by a nuclear translocation of p50 and p65 NF-kappaB subunits and their binding to NF-kappaB sites of hBD-2 promoter region. With site-directed mutagenesis, we demonstrated the requirement of two proximal NF-kappaB binding sites (pkappaB1, -205 to -186; pkappaB2, -596 to -572) but not the distal site (dkappaB, -2193 to -2182) in supporting IL-17A-induced hBD-2 promoter activity. These results are consistent with the data of the chromatin immunoprecipitation assay, which showed enhanced p50 binding to these pkappaB sites but not the dkappaB site in cells after IL-17A treatment. We also found that the NF-kappaB binding cofactor, IkappaB-zeta, was up-regulated by IL-17A, and the knockdown of IkappaB-zeta significantly diminished the IL-17A-induced hBD-2 expression. This is the first demonstration of the involvement of two proximal NF-kappaB sites and IkappaB-zeta in the regulation of hBD-2 by IL-17A, two important genes responsible for host defense.     

Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell

The mechanisms for lactic acid bacteria (LAB) to inhibit Salmonella invasion appear to be multifactorial and include the adhesion of LAB to host intestine epithelium, the production of organic acids, or bacteriocin by LAB cells. Previously, we found a strain of Lactobacillus acidophilus isolated from swine, i.e. strain LAP5, was with antagonistic effect against Salmonella typhimurium. This strain LAP5 was also found to meet the requirements for probiotic use. In this study, we evaluate the potential of LAP5 strain to protect the human or swine from infection by Salmonella choleraesuis. We present evidence that the culture of LAP5 was able to inhibit the invasion of S. choleraesuis to human Caco-2 cell line. The LAP5 cell culture showed a higher inhibitory effect on the invasion of S. choleraesuis to Caco-2 cells than the spent culture supernatant (SCS) of LAP5 did. Also, the pH, organic acids or the bacteriocin, which act at low pH conditions, may play the role of antagonistic effect. The addition, adhesion of LAP5 cells to Caco-2 cell line may also play roles to reduce the invasion of S. choleraesuis.