Interleukin-8 secretion by epithelial cells infected with diffusely adherent Escherichia coli possessing Afa adhesin-coding genes

Escherichia coli that adhere sparsely to human epithelial (HEp-2) cells are known as diffusely adherent E. coli(DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)-identified originally as a uropathogenic factor-in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL-8 in cultures of human carcinoma epithelial cells (e.g., HEp-2, Caco-2, and T84). Nineteen afa-positive DAEC strains were examined for their ability to induce IL-8 secretion, and only 7 strains (37%; 7/19) induced IL-8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL-8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.     

Interleukin‐8 induction due to diffusely adherent Escherichia coli possessing Afa/Dr genes depends on flagella and epithelial Toll‐like receptor 5

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL‐8 secretion in cultures of human carcinoma epithelial cells (HEp‐2, Caco‐2), is enterovirulent. In the present study, we examined whether IL‐8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL‐8 high‐inducing strains were highly motile in swarming tests. Partially purified flagella induced IL‐8 in a dose‐dependent manner. However, IL‐8 induction was inhibited by small‐interfering RNA against TLR5 or by treating flagella with disialoganglioside‐GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL‐8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL‐8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL‐8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL‐8 secretion.     

Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli

Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity.     

Diffusely adherent Escherichia coli strains expressing Afa/Dr adhesins (Afa/Dr DAEC): hitherto unrecognized pathogens

Diffusely adherent Escherichia coli (DAEC) strains are currently considered to constitute a putative sixth group of diarrheagenic E. coli. However, on the basis of their diffuse adherence to HEp-2 and HeLa cells, the detection of afa/dra/daa-related operons encoding this adherence phenotype, and the mobilization of decay-accelerating factor, both commensal and pathogenic strains can be classified as Afa/Dr DAEC isolates. Furthermore, strains associated with diarrheal diseases and strains causing extra-intestinal infections can also be identified as Afa/Dr DAEC strains. Although several cell signaling events that occur after epithelial cells have been infected by Afa/Dr DAEC have been reported, the pathophysiological processes that allow intestinal and extra-intestinal infections to develop are not fully understood. This review focuses on the genetic organization of the afa/dra/daa-related operons and on the virulence factors that trigger cellular responses, some of which are deleterious for the host cells. Finally, this review suggests future lines of research that could help to elucidate these questions.     

The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells

Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).     

Norepinephrine-dependently released Dr fimbriae of diffusely adhering Escherichia coli strain IH11128 promotes a mitogen-activated protein kinase ERK1/2-dependent production of pro-inflammatory cytokine, IL-8 in human intestinal Caco-2/TC7 cells

The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.     

Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC)

Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.     

Vaginal Lactobacillus isolates inhibit uropathogenic Escherichia coli

The purpose of this study was to investigate the antibacterial activities of Lactobacillus jensenii KS119.1 and KS121.1, and Lactobacillus gasserii KS120.1 and KS124.3 strains isolated from the vaginal microflora of healthy women, against uropathogenic, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strains IH11128 and 7372 involved in recurrent cystitis. We observed that some of the Lactobacillus isolates inhibited the growth and decreased the viability of E. coli IH11128 and 7372. In addition, we observed that adhering Lactobacillus strains inhibited adhesion of E. coli IH11128 onto HeLa cells, and inhibited internalization of E. coli IH11128 within HeLa cells.     

Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli

Background

Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn''s disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology

VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal Findings

C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions

Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.     

hCEACAM1-4L downregulates hDAF-associated signalling after being recognized by the Dr adhesin of diffusely adhering Escherichia coli

Human decay accelerating factor (hDAF, CD55) and members of the c arcino e mbryonic- a ntigen-related c ell- a dhesion m olecules (hCEACAMs) family are recognized as receptors by Gram-negative, diffusely adhering Escherichia coli (DAEC) strains expressing Afa/Dr adhesins. We report here that hCEACAM1-4L has a key function in downregulating the protein tyrosine Src kinase associated with hDAF signalling. After infecting HeLa epithelial cells stably transfected with hCEACAM1-4L cDNA with Dr adhesin-positive E. coli , the amount of the pTyr⁴¹⁶-active form of the Src protein decreased, whereas that of the pTyr⁵²⁷-inactive form of Src protein did not increase. This downregulation of the Src protein implies that part of the hCEACAM1-4L protein had been translocated into lipid rafts, the protein was phosphorylated at Tyr residues in the cytoplasmic domain, and it was physically associated with the protein tyrosine phosphatase, SHP-2. Finally, we found that the hCEACAM1-4L-associated SHP-2 was not phosphorylated and lacked phosphatase activity, suggesting that the downregulation of Src protein associated with hDAF signalling results from the absence of dephosphorylation of the pTyr⁵²⁷-inactive form necessary for Src kinase activation.     

Mycobacterial mammalian cell entry protein 1A (Mce1A)-mediated adherence enhances the chemokine production by A549 alveolar epithelial cells

Mycobacterial mammalian cell entry protein 1A (Mce1A) is involved in the uptake of bacteria in non-phagocytic cells and also possibly in granuloma formation. However, it has not been clarified whether the interaction between mycobacterial Mce1A and epithelial cell induces chemokine and cytokine production which is required for granuloma formation. To this end, we infected A549 alveolar epithelial cells in vitro with E. coli expressing Mce1A on the cell surface and examined the resultant chemokine/cytokine production. Mce1A promoted bacterial adherence and internalization of E. coli into A549 cells, and these recombinant bacteria induced high levels of MCP-1 and IL-8 production, compared to E. coli harboring the plasmid vector alone. Chemokine production was enhanced by the internalization of recombinant E. coli expressing Mce1A because cytochalasin D treatment partially inhibited MCP-1 and IL-8 production. However, Mce1A-coated latex beads did not induce the chemokine production. These results suggest that although Mce1A does not induce production of chemokines, it may promote chemokine induction by augmenting the interaction between bacteria and epithelial cells.     

Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells

Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.     

Apical expression of human full-length hCEACAM1-4L protein renders the Madin Darby Canine Kidney cells responsive to lipopolysaccharide leading to TLR4-dependent Erk1/2 and p38 MAPK signalling

CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.     

Covert operations of uropathogenic Escherichia coli within the urinary tract

Entry into host cells is required for many bacterial pathogens to effectively disseminate within a host, avoid immune detection and cause disease. In recent years, many ostensibly extracellular bacteria have been shown to act as opportunistic intracellular pathogens. Among these are strains of uropathogenic Escherichia coli (UPEC), the primary causative agents of urinary tract infections (UTIs). UPEC are able to transiently invade, survive and multiply within the host cells and tissues constituting the urinary tract. Invasion of host cells by UPEC is promoted independently by distinct virulence factors, including cytotoxic necrotizing factor, Afa/Dr adhesins, and type 1 pili. Here we review the diverse mechanisms and consequences of host cell invasion by UPEC, focusing also on the impact of these processes on the persistence and recurrence of UTIs.     

Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes

Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.     

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.     

HIF‐1α mediates the induction of IL‐8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT‐like behaviour

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro‐angiogenic and pro‐inflammatory genes. Here, we demonstrate that exposure of cryptic‐like intestinal epithelial cells to C1845 bacteria induces HIF‐1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane‐associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI‐3K decreases HIF‐1α protein levels and blocks C1845‐induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria‐induced HIF‐1α regulates the expression of IL‐8, VEGF and Twist1, thereby pointing to a role for HIF‐1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E‐cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition‐like phenotype. Since HIF‐1α silencing results in reversion of bacteria‐induced EMT markers, we speculate that HIF‐1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.     

Localization of TLR2 and MyD88 to Chlamydia trachomatis inclusions. Evidence for signaling by intracellular TLR2 during infection with an obligate intracellular pathogen

Chlamydia trachomatis is an obligate intracellular gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis.We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.     

Interleukin-8 secretion of human epithelial and monocytic cell lines induced by middle ear pathogens

Otitis media with effusion (OME) is one of the most common diseases in children. Alloiococcus otitidis, a new gram-positive bacterial species, was isolated from the middle ear fluid of children with OME; however, the pathogenic role of this bacteria is yet unknown. In this study, the ability of cultured epithelial cell lines (Hep-2 and Hela) and monocytic cell lines (THP-1 and U 937) to secrete chemokine interleukin-8 (IL-8) in response to the A. otitidis organism and three bacterial organisms mainly detected from middle ear fluid in OME, and bacterial cell components was investigated. When stimulated with four viable bacterial cells, epithelial cells and monocytes secreted IL-8 in a time-dependent manner. The monocytes produced significantly higher levels of IL-8 than the epithelial cells. Compared with that by viable bacterial cells, IL-8 secretion by stimulated epithelial cells and monocytes was reduced when the bacteria were heated and treated with glutaraldehyde. With bacterial stimulations, cell treatment of interferon-gamma caused monocytes to increase the induction of IL-8 production, however, the induction of monocyte differentiation caused monocytes to reduce the induction of IL-8 production. Furthermore, epithelial cells and monocytes stimulated by four viable bacterial organisms physically separated from cultured cells reduced the induction of IL-8 compared with directly stimulated cells, and monocytes stimulated with soluble extracts prepared from A. otitidis organisms produced IL-8 in a dose-dependent manner. These results suggest that part of the IL-8 stimulation of the A. otitidis organism may exist in a diffusable factor released by the bacteria or soluble components of the bacteria itself.     

Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells

Approximately 60% of Helicobacter pylori strains are cagA ⁺ and this genotype is more frequently associated with duodenal ulcer disease. Although most wild-type cagA ⁺ strains are both cytotoxigenic and induce enhanced Interleukin-8 (IL-8) secretion in gastric epithelial cells, isogenic cagA ⁻ mutants retain full activity in these assays; thus, cagA appears to be a marker of enhanced virulence. Delineation of the nucleotide sequence of a 4 kb region upstream of cagA allowed the identification of 966 bp ( picA ) and 2655 bp ( picB ) open reading frames encoding 36 kDa and 101 kDa polypeptides, respectively. picA and picB constitute an operon in opposite orientation to cagA . The deduced picB product showed significant homology (26% identity and 50% similarity) with the Bordetella pertussis toxin secretion protein (PtlC). Of 55 H. pylori clinical isolates, the picA and picB segment was conserved exclusively in cagA ⁺ strains and present in all isolates from patients with duodenal ulceration, versus 59% of isolates from patients with gastritis alone ( P =0.01). Using gene-replacement techniques, we constructed picA and picB mutant H. pylori strains and demonstrated that the picB gene product is involved in the induction of IL-8 expression in gastric epithelial cells. Further, Northern blot hybridization and RT-PCR data showed that picA and picB are co-transcribed and an insertional mutation in picA ablates picB expression. These studies indicate a role of picA and picB in the induction of an inflammatory response in gastric epithelial cells either directly or by enabling secretion of an unidentified product, and suggest a mechanism for the overrepresentation of strains possessing these genes in patients with peptic ulceration.