Outer Membrane Protein A of Escherichia coli O157:H7 Stimulates Dendritic Cell Activation
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感染与免疫杂志 2006年第5期
Departments of Microbiology and Immunology Pathology
Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555-1070
ABSTRACT
Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.
INTRODUCTION
Enterohemorrhagic Escherichia coli (EHEC) strains are a class of pathogenic microorganisms responsible for numerous food- and waterborne outbreaks and can cause illness ranging from nonbloody diarrhea to copious bloody discharge in humans. In some individuals, the disease progresses to a more serious stage, and about 2 to 7% of the diarrheal cases (particularly in the pediatric population) can be fatal due to acute kidney failure (hemolytic-uremic syndrome [HUS]) (reviewed in references 26 and 28). Strains of EHEC O157:H7, the most common EHEC serotype in North America, colonize the intestine and produce multiple determinants which cause the pathology associated with the disease, with Shiga toxin (Stx) being a key feature of virulence.
Stx produced by EHEC strains are responsible for severe damage to epithelial and endothelial cells in the intestinal tract (26, 28) and in the kidneys (reviewed in reference 25), but the sequential events leading to HUS are largely unknown. It has been proposed that Stx-mediated activation of immune cells in the gut initiates the pathology of HUS in the kidneys (11); however, it is unclear whether this process could occur alone or in synergy with other bacterial components. While EHEC O157:H7 can produce a distinct lesion known as the attaching and effacing lesion (24, 49, 51), several proteins, including Iha, Cah, ToxB, OmpA (outer membrane protein A), and the long polar fimbriae, have been identified as putative adhesion factors, facilitating attachment of EHEC to host intestinal epithelial cells. The precise roles of these other factors in disease pathogenesis have not yet been defined (46, 47, 50).
OmpA is one of the major proteins in the membrane of E. coli and is highly conserved among gram-negative bacteria (7). It plays a multifunctional role in the biology of the bacteria, acting as a phage and colicin receptor, serving as a mediator in F-factor-dependent conjugation (35), and maintaining structural integrity of membranes and generation of normal cell shape (40). In E. coli K1 strains, OmpA is also known to be associated with the pathogenesis of neonatal meningitis because it mediates invasion of E. coli K1 to brain microvascular endothelial cells (31) and plays a key role during the initial processes of bacterial adhesion and invasion (30, 31, 37).
More recently, the role of OmpA in adherence was established during the screening of an EHEC O157:H7 transposon insertion mutant library for hyperadherent mutants (48). A mutation of the tcdA gene, encoding a transcriptional activator associated with L-threonine transport and degradation, resulted in elevated expression of OmpA and hyperadherence of EHEC O157:H7 to HeLa and Caco-2 cells (48). Inactivation of ompA in the tdcA mutant abolished the hyperadherent phenotype, and mutation of ompA alone in the wild-type EHEC O157:H7 strain reduced adherence by 13.5%. Furthermore, OmpA-specific antiserum inhibited the adherence of other EHEC O157:H7 strains to HeLa cells. These studies collectively imply an important role for OmpA in mediating adhesion of EHEC O157:H7 strains to host cells (48).
It has been shown that antigen-presenting cells (APCs) can recognize Klebsiella pneumoniae OmpA and are activated by this interaction (16, 41). K. pneumoniae OmpA induces the expression of costimulatory molecules and CD83 on human dendritic cells (DCs) and triggers cytokine production by murine and human macrophages (13, 15). OmpA appears to possess a new type of pathogen-associated molecular pattern (PAMP) because it is conserved among the Enterobacteriaceae family, is essential for bacterial survival and virulence, and can activate APCs (14, 29).
DCs are potent APCs with the unique ability to activate nave T cells (reviewed in reference 23). DCs are widely distributed in the skin and the mucosal tissues in the intestinal tract and airways. Upon recognizing PAMPs via specialized receptors such as the C-type lectin, Fc receptors, and Toll-like receptors (TLRs), DCs detect the presence of bacteria in the host tissues, capturing them or their products, migrate to the T-cell areas in lymph nodes, and activate nave T cells (reviewed in reference 23). During these processes, DCs become mature and are activated to produce diverse cytokines. Over the past few years, much has been learned about the biology of EHEC O157:H7 strains, but it is not known whether these bacteria and their products interact with DCs. It is possible that OmpA of EHEC O157:H7 can function as a PAMP to stimulate DC cytokine production and migration. To test this hypothesis, we generated recombinant OmpA proteins, as well as EHEC O157:H7 strains that differ in expression level of OmpA. Using both murine and human DC systems, we demonstrated in this study that purified OmpA proteins and bacteria expressing high levels of OmpA were efficient stimuli for DCs to produce cytokines and that DC cytokine production was significantly enhanced by the bacterium-antibody (Ab) immune complex. In addition, we provided evidence for bacterially induced migration of DCs through polarized colonic epithelial cells.
MATERIALS AND METHODS
Bacterial strains, plasmids, and reagents. Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were routinely grown in Luria-Bertani (LB) broth or on L agar at 37°C overnight (21). When indicated, the strains were grown in Dulbecco's modified Eagle's medium (Cell Growth/Mediatech, Inc.). Antibiotics were added to LB media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 30 μg/ml; streptomycin, 100 μg/ml; and nalidixic acid, 15 μg/ml. Lipopolysaccharide (LPS) (Salmonella enterica serovar Typhimurium) was purchased from Sigma (St. Louis, MO).
Recombinant DNA techniques and plasmid construction. Plasmid DNA was isolated from overnight bacterial cultures by using a QIAGEN QIAprep plasmid preparation kit and introduced by transformation of RbCl2-competent E. coli DH5 strains or by electroporation of EHEC O157:H7 as described by Dower et al. (8). Standard molecular methods were used for restriction endonuclease analyses, ligation, and construction of recombinant plasmids (21). The ompA gene was amplified from EHEC O157:H7 strain EDL933 by PCR using the primer pair 5'-CCGATATCGGTAGAGTTAATATTGA-3' and 5'-CCTCTAGAAAGCGGTTGGAAATGGAAG-3' (the underlining indicates EcoRV and XbaI sites). The amplicon was digested with EcoRV and XbaI and cloned in the corresponding restriction sites of pACYC184 to create pOmpA.
Excision of the cat cassette from the EHEC 86-24 ompA mutant strain AGT601. Strain AGT601 (48) was created previously by inactivation of the ompA gene with a cat cassette (6). To create the AGT601S strain for this study, the cat gene was excised by FLP recombination, using the temperature-sensitive replication plasmid pCP20, following the protocol previously described (6).
Western blot analysis. Overnight bacterial cultures were adjusted into portions of 2.0 x 108 bacteria, washed in phosphate-buffered saline (pH 7.4), resuspended in 200 μl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer, and boiled for 10 min. Solubilized proteins were separated on 12% SDS-PAGE gels according to the method of Laemmli (19), and proteins were transferred to Immobilon-P membranes (Millipore) by using a Trans-Blot SD transfer cell (Bio-Rad). After nonspecific sites were blocked, the membrane was incubated with rabbit anti-OmpA antibody (1:30,000) and then with goat anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (1:30,000; Sigma) and developed. The OmpA antibodies used in this study have been purified and their specificity previously reported (31).
Expression and purification of the His-tagged OmpA protein. DNA fragments containing the complete open reading frame of ompA from EHEC O157:H7 strain EDL933 were obtained using PCR and the following primers: 5'-CCGAATTCATGAAAAAGACAGCTATCGCGA-3' and 5'-AACTAAGCTTTGCGGCTGAGTTACAACGTC-3' (underlining indicates EcoRI and HindIII sites). The amplicons were cloned into pBad/MycHis (Invitrogen), and the resulting plasmid (pBadOmpA) was introduced into E. coli TOP10 cells. Expression of the gene was induced by the addition of arabinose to a final concentration of 0.2% and incubation at 37°C for 4 h. The His-tagged OmpA protein was purified following protocols recommended by the manufacturers of the ProBond purification system (Invitrogen). The amount of purified protein was quantified by a standard Bradford assay (Bio-Rad) (3a). A Limulus amebocyte lysate test indicated that the different stocks of purified OmpA samples contained 0.86 to 1.6 μg/ml of LPS. To obtain endotoxin-free His-tagged OmpA protein, the purified protein was passed through a Detoxigel Affinity Pak prepacked column (Pierce). However, the yield of OmpA after the elution was only 1/10 of the original concentration (data not shown). Therefore, the experiments were performed in the presence of polymyxin B, as described below.
Generation of BM-DCs. C57BL/6 and C3H/HeJ mice were purchased from Jackson Laboratory (Bar Harbor, ME). A spontaneous mutation in Toll-like receptor 4 (TLR4) occurring at the LPS response locus makes C3H/HeJ mice endotoxin resistant. All mice were maintained under specific-pathogen-free conditions and used for experiments at 6 to 10 weeks of age with protocols approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX). Bone marrow-derived DCs (BM-DCs) were generated as previously described (33), with certain modifications. Briefly, bone marrow cells were cultured in petri dish plates (Fisher Scientific, Houston, TX) at 2 x 106 cells per 10 ml of 10% fetal bovine serum-supplemented Iscove's modified Dulbecco's medium (Invitrogen/Gibco). Culture supernatants of J558L cells that had been transfected with the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene were used as the source of GM-CSF. Nonadherent cells were harvested at day 8 and further cultured in a six-well plate overnight. Resultant nonadherent cells were typically >80% CD11c+ cells as judged by flow cytometric analysis. Sometimes, CD11c+ cells were directly purified from a day-8 culture to >95% purity with microbeads according to the manufacturer's protocol (Miltenyi Biotec, Auburn, CA).
Stimulation of murine DCs. Harvested BM-DCs were washed, suspended in complete Iscove's modified Dulbecco's medium, and stimulated with different concentrations of LPS (0.04, 0.08, or 0.16 μg/ml), purified His-tagged OmpA (0.1, 0.25, 0.35, 0.5, 0.7, or 1.05 μg/ml), or different strains of bacteria at 1.25 x 105 cells/200 μl/well in 96-well plates or 6.25 x 105 cells/0.5 ml/well in 24-well plates. The stimulation culture was not supplemented with any cytokines, including GM-CSF. For some experiments, bacteria were preincubated with anti-OmpA polyclonal Ab (PAb) or monoclonal Ab (MAb) (kindly provided by N. V. Prasadarao and S. P. Singh [39], respectively) before being added to DC cultures. Supernatants were harvested at 24 h for measuring levels of interleukin-1 (IL-1), IL-1, IL-10, and IL-12p40 (see below). To assay the effect of LPS on DC stimulation, samples of His-tagged OmpA or LPS were preincubated for 10 min with 10 μg/ml polymyxin B sulfate (Sigma) before addition to the cells. Supernatants were harvested at 24 h for measuring levels of IL-10 and IL-12p40.
Purification of human DCs. DCs were purified from healthy-human peripheral blood mononuclear cells by negative selection using the magnetic column separation system (StemCell Technologies, Inc.). Monocytes (5 x 105/ml) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, GM-CSF (100 ng/ml), and IL-4 (50 ng/ml) in 24-well plates. Cytokines were replenished every 3 days. Nonadherent, immature DCs were obtained at 7 days, and only immature CD11a+ CD83– DCs were used in the experiments listed below.
Polarized intestinal epithelial cell model. Caco-2 cells, a human adenocarcinoma cell line (American Type Culture Collection, Bethesda, MD), were seeded on membrane inserts in Costar 12-well tissue culture transwell plates (0.4- or 3-μm filters; Corning, Inc., Corning, NY) until confluent. This cell line was used between passages 5 and 15 and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 110 mg/liter of sodium pyruvate, 100,000 IU/liter of penicillin, and 100 mg/liter of streptomycin (Gibco BRL, Grand Island, NY) at 37°C in 5% CO2. The cells were allowed to grow into a confluent, differentiated, polarized monolayer, establishing microvilli, brush borders, and tight junctions.
DC transmigration assay using polarized Caco-2 cells. Caco-2 cells grown on 3-μm filter inserts were infected on the apical (AP) surface with 1 x 107 CFU/ml of EHEC O157:H7 strain 86-24, AGT601S, or AGT601S transformed with a plasmid that carries the ompA gene [AGT601S(pOmpA)] for 1 h at 37°C and 5% CO2. After the prestimulation time, freshly isolated human DCs (1 x 105) were added to the basolateral (BL) chamber and migration was assessed at 4 h. Basolateral DCs with medium only added to the AP chamber served as a negative control. The AP and BL supernatants were collected for counting in a hemacytometer to determine immune-cell migration. Additionally, AP and BL supernatants from DC migration were analyzed for cytokine secretion.
Bacterial-adhesion assays. Caco-2 cells were seeded with 1 x 105 cells/well and incubated for 48 h at 37°C and 5% CO2 in 24-well plates (Corning), and an adhesion assay was performed as described previously (48). Briefly, cell monolayers were washed twice with phosphate-buffered saline and then infected with 1 x 107 bacteria for 4 h or 5 h. Adherence was evaluated qualitatively by Giemsa staining and quantitatively by plating adherent bacteria on L agar plates with the proper antibiotic. In the DC transmigration assay and following immune-cell counting, adherence of bacteria to the AP surface of the polarized cells was evaluated quantitatively by plating serial dilutions of the bacteria, recovered by Triton X-100 treatment (48), on L agar plates with the appropriate antibiotic and growing overnight at 37°C.
Murine and human cytokine assays. To measure the levels of IL-1, IL-1, IL-10, and IL-12p70 in murine DC cultures, specific enzyme-linked immunosorbent assays (ELISAs) were performed with OptEIA kits (BD Biosciences) as previously described (17). For the experiments with human DCs, supernatants were collected, at 5 h after bacterial infection and immune-cell migration, from the AP and BL surfaces of Caco-2 polarized cells. The amount of human cytokine secretion in the supernatants was quantified by a Bio-Rad BioPlex multiplex cytokine system, and this process was performed at the GI Immunology Core facility (UTMB).
Statistical analysis. Statistical differences during bacterial adhesion to epithelial cells were determined by a paired t test. Similarly, to determine the differences in cytokine production by different stimuli, a paired t test was also used.
RESULTS
EHEC OmpA induced DCs to secrete cytokines in a dose-dependent and TLR4-independent manner. DCs discriminate between different microbial pathogens and induce T-cell responses (32). However, it is not clear to what extent EHEC O157:H7 recognition (whole organism or individual microbial components) could induce different functional states of DCs that favor different T-cell effector phenotypes. We examined whether coculturing DCs with purified OmpA induced maturation and specific cytokines known to be secreted by DCs (32). Due to the difficulty in purifying OmpA without contamination by lipoprotein (or endotoxin [see Materials and Methods]), we used DCs derived from C3H/HeJ mice and examined DC cytokine production in response to purified EHEC OmpA and LPS (a negative control). As shown in Fig. 1A through D, OmpA induced DCs to produce high levels of IL-1, IL-1, IL-10, and IL-12p70 in a dose-dependent fashion, while untreated DCs (control) did not produce measurable levels of cytokines. Since exogenous LPS (even at 0.16 μg/ml) did not stimulate DCs to produce these cytokines, these results suggest that purified OmpA activated DCs via a TLR4-independent mechanism.
To further determine whether DC cytokine production was due to OmpA and not to LPS contamination, we generated DCs from C57BL/6 mice and examined DC cytokine production in response to purified EHEC OmpA and LPS in the presence or absence of polymyxin B. Figure 1E and F show that there was a dose-dependent increase in IL-12p70 and IL-10 production in OmpA-stimulated DCs, even in the presence of polymyxin B. In contrast, addition of polymyxin B to the LPS control samples totally abolished cytokine production. Taken together, our results suggested that DC responses were mediated in part via TLR4-independent mechanisms due to OmpA stimulation and in part via TLR4-dependent mechanisms (due to trace amounts of LPS).
EHEC O157:H7 strains with different expression levels of OmpA were capable of stimulating DCs to produce cytokines. We have previously shown that OmpA acts as an adhesin facilitating binding of EHEC O157:H7 to intestinal epithelial cells (48). However, it was unclear in that study whether the adherent phenotype of the ompA mutant could be rescued in trans by complementation with the ompA gene on a plasmid. To test this possibility and to examine the role of OmpA in bacterium- DC interaction, we transformed EHEC strain AGT601S with a plasmid that carries the ompA gene, which is referred to as AGT601S(pOmpA) in this study. Adhesion assays performed at two different time points indicated that EHEC mutant strains AGT601 (ompA::cat) and AGT601S (ompA mutant) adhered to Caco-2 cells at a level comparable to that of our previous findings with HeLa cells (48) but that they adhered to a lower extent (50% lower) than the wild-type 86-24 strain (Table 2). At 5 h postinfection, there was not a statistical difference between the ompA mutants and the wild-type control, probably due to the expression of other adhesins, such as intimin. In contrast, the adherence of strain AGT601S(pOmpA) at 4 h postinfection was significantly higher than that of the wild-type control (Table 2) (P < 0.05). Western blot analysis further confirmed the expression levels of OmpA protein in the different strains (Fig. 2A). While no OmpA expression was detected in strain AGT601S (Fig. 2A, lane 2), it appeared that that strain AGT601S(pOmpA) (Fig. 2A, lane 3) expressed slightly more OmpA protein than did the wild-type strain (Fig. 2A, lane 1). Collectively, these results confirmed and extended our previous findings (48), indicating a role for OmpA in adherence of EHEC to the host cells.
To examine whether DCs responded to these bacterial strains differently, we incubated BM-DCs of C3H/HeJ mice with increasing concentrations of EHEC strain 86-24, AGT601S, AGT601S (pACYC184), or AGT601S(pOmpA) or the E. coli K-12 strain MG1655 (nonpathogenic, OmpA-positive control strain) and measured cytokine production at 24 h postinfection. As shown in Fig. 3, no secretion of cytokines was detected when DCs were left untreated or incubated with bacteria at a bacterium-to-cell ratio of 100:1. At the 500:1 and 1,000:1 ratios, DCs produced high levels of IL-1, IL-1, IL-10, and IL-12p70. DCs exposed to strain AGT601S(pOmpA) consistently produced significantly higher levels of IL-1, IL-10, and IL-12p70 than did the wild-type controls. The cytokine production induced by the complemented ompA mutant strain was not attributed to the presence of the empty vector pACYC184. Although we observed a marginal reduction in expression of IL-1 and IL-10 with the ompA mutant groups, especially with the low-bacterial-dose groups, this trend was not consistent in all of the independent repeats. Finally, we tested whether any OmpA-positive E. coli strain was capable of stimulating cytokine production when DCs were treated with bacterium-to-cell ratios similar to those used with the EHEC strains. Surprisingly, E. coli K-12 strain MG1655 induced DCs to produce low levels of IL-1, IL-10, and IL-12p70 only at the ratio of 1,000:1. Taken together, these studies suggest that overexpression of OmpA on a plasmid was associated with an increase in DC cytokine secretion and that not all OmpA-positive strains were capable of inducing DCs to respond to bacterial stimulus.
Anti-OmpA Abs significantly enhance DC cytokine secretion. We have previously shown that OmpA-specific antiserum inhibited the adherence of different EHEC O157:H7 strains to HeLa cells by approximately 25% (48). We hypothesized that incubation of bacteria with anti-OmpA sera prior to infection would block interaction of OmpA with DCs and therefore reduce DC cytokine secretion. To test this hypothesis, we focused on the strain AGT601S(pOmpA) and used serial dilutions of rabbit anti-OmpA PAb, mouse anti-OmpA MAb (IgG2a), and an isotype control (IgG2a). Surprisingly, we found that in comparison to results for the groups with untreated bacteria, preincubation of bacteria with OmpA-specific Abs (particularly at a 10:1 or a 1:1 dilution of the Ab) significantly increased the production of IL-1, IL-1, and IL-10 (Fig. 4). Preincubation of bacteria with an isotype control also enhanced the production of IL-1 and IL-1 (but not IL-10), but the levels of induction were significantly lower than those produced by OmpA-specific antisera. To examine whether anti-OmpA Ab alone had direct effects on DC cytokine production, we left cells untreated or treated them with serial dilutions of anti-OmpA MAb alone or MAb-preincubated strain AGT601S(pOmpA). It was evident in Fig. 5 that MAb alone was incapable of stimulating DCs to produce cytokines and that the bacterium-MAb immune complex was a potent stimulus for DCs to produce high levels of IL-1, IL-1, and IL-10.
Polarized Caco-2 cells exposed to EHEC O157:H7 were capable of producing cytokines and triggering DC migration. DC activation and migration are essential processes for the functionality of DCs (23). Having demonstrated a role for EHEC OmpA in DC cytokine secretion, we then tested whether EHEC O157:H7, upon binding to host cells, can stimulate the migration of DCs across intestinal epithelial cells. For these experiments, the AP surface of polarized Caco-2 cells was infected with EHEC strains for 1 h, prior to the addition of DCs in the BL chamber. After an additional 4 h of incubation, the AP and BL cell supernatants were collected and analyzed for adherent bacteria, Caco-2 cell cytokine secretion, and DC migration. As shown in Fig. 6A, at 5 h postinfection, we recovered more bacteria of the wild-type EHEC O157:H7 strain 86-24 from the AP surface of Caco-2 cells than we recovered of the ompA mutant strain (P < 0.05). When the ompA mutant was complemented in trans with the pOmpA plasmid, the levels of adhesion were regained; however, they did not fully restore the wild-type phenotype. These transwell filter results were comparable with those obtained with the Caco-2 monolayers (Table 2), suggesting a role for OmpA in facilitating the binding of bacteria to the apical portion of Caco-2 cells.
To determine whether binding of EHEC strains to the AP and BL surfaces of Caco-2 cells could induce cells to produce cytokines, we collected culture supernatants from both chambers at 5 h postinfection and measured cytokine production. It was evident that the binding of wild-type and ompA mutant strains to the AP surface of Caco-2 cells induced the secretion of several cytokines and chemokines, including proinflammatory cytokines (IL-8, CCL2/monocyte chemoattractant protein [MCP-1], and CCL4/macrophage inflammatory protein 1 [MIP-1]), those that augment neutrophil survival (granulocyte colony-stimulating factor and GM-CSF), those that cause activation of cells from circulation (IL-6), and those that induce production of more cytokines (tumor necrosis factor alpha [TNF-]). Furthermore, cytokine production was more prominent in the BL supernatants (Table 3). Basal levels of cytokine secretion were detected when the polarized Caco-2 cells were incubated with DCs in the absence of bacterial stimuli (Table 3); however, they were much lower than levels for those cells stimulated with bacteria. Furthermore, infection of Caco-2 cells with the different bacterial strains, but in the absence of DCs, confirmed that cytokines detected were produced by the intestinal epithelial cells in response to the bacterial stimuli (data not shown). However, we did not observe major differences in cytokine secretion between the wild-type strain and the ompA mutant, regardless of their ability to bind Caco-2 cells, suggesting the possible involvement of bacterial factors other than OmpA in cytokine induction.
Given the broad profile of cytokine production by Caco-2 cells, we reasoned whether binding of EHEC to Caco-2 cells promoted migration of DCs from the BL side to the AP side. We counted the percentage of DCs recovered from each of the two chambers and showed that binding of the wild-type EHEC strain 86-24, its ompA mutant (AGT601S), or the complemented strain AGT601S(pOmpA) to the AP surface of Caco-2 cells promoted DC migration (Fig. 6B). Surprisingly, strain AGT601S was more efficient than the wild-type strain in promoting DC migration to the AP side (P < 0.01) (Fig. 6B). This phenotype was reversible upon introduction of the pOmpA plasmid (Fig. 6B). These data suggest that in the absence of the OmpA protein, other bacterial surface proteins were involved in stimulating DC migration.
DISCUSSION
EHEC O157:H7 strains are known to be noninvasive organisms, to adhere to the apical surface of colon epithelial cells, and to cause infection that is characterized by acute inflammation of the colonic mucosa (26). EHEC is thought to signal colon epithelial cells to produce proinflammatory signals that can chemoattract and activate leukocytes and activate mucosal inflammation (43, 44). Stx and flagellin are examples of EHEC virulence factors that have been reported to stimulate the production of proinflammatory chemokines, resulting in an influx of neutrophils and other leukocytes in cultured human colon cell lines (3, 34, 43, 44). Further, we have recently demonstrated that the major functional signaling pathway for the up-regulated production of proinflammatory chemokines by EHEC-infected human colon epithelium in vivo involves H7 flagellin signaling through TLR5, where Stx signaling is not required (22). No additional data as to whether any other EHEC factor(s) is associated with the recruitment and/or activation of DCs, potent APCs that play a crucial role for host defense against invading pathogens (27), are yet available. We reported in this study that the OmpA protein from EHEC O157:H7 stimulated DC maturation and secretion of cytokines, which is not observed with the nonpathogenic E. coli OmpA-positive strain MG1655. Binding of OmpA-expressing EHEC O157:H7 to the apical surface of polarized Caco-2 cells is not sufficient to stimulate secretion of proinflammatory cytokines and transmigration of DCs, which suggests that other EHEC O157:H7 surface components in addition to OmpA are involved in stimulating DC migration.
Host defense against bacteria is dependent upon clearance by phagocytes (1). Several reports by Jeannin and colleagues have demonstrated that K. pneumoniae OmpA up-regulated the secretion of cytokines (e.g., TNF-, IL-1, IL-10, and IL-12) from murine and human macrophages and bone marrow-derived or purified DCs (4, 15). These in vitro data collectively suggest that OmpA, by up-regulating cytokine production, may favor in vivo leukocyte recruitment to the site of infection and leukocyte activation. In support of this hypothesis, it has recently been demonstrated that subcutaneous injection of K. pneumoniae OmpA in mice triggered a local and transient inflammation accompanied by an increase in draining lymph node cell numbers, with a higher proportion of DCs and macrophages (15). Macrophages are recruited to kill bacteria, and the interaction between OmpA and macrophages initiates a pathway by which innate cells respond to and eliminate pathogens. More-complex cellular and humoral responses to K. pneumoniae OmpA have been suggested by the findings that K. pneumoniae OmpA is recognized by cellular receptors such as LOX-1 and SREC-1 for bacterial binding and is recognized by TLR2 for signaling (13). Therefore, the sequential events include the initial interaction between cellular receptors and K. pneumoniae OmpA, the activation of the proinflammatory signal pathway (including the elevation of humoral PTX3), and the enhanced binding of PTX3 to K. pneumoniae OmpA. Cellular and humoral (PTX3-mediated) recognition are complementary in mediating the innate response to K. pneumoniae OmpA (13).
We have shown in this study that recombinant OmpA of EHEC stimulated murine DCs to produce IL-1, IL-1, IL-10, and IL-12p70 in a dose-dependent and TLR4-independent manner (Fig. 1). At this stage, the receptor(s) that mediates these cytokine responses is unclear. In an attempt to understand the relative contribution of OmpA in DC responses to EHEC bacteria, we examined the DC cytokine responses to EHEC O157:H7 strain 86-24, its isogenic ompA mutant (AGT601S), and the complemented strain AGT601S(pOmpA) and obtained two interesting findings. First, murine DCs responded to bacterial stimulation by producing high levels of IL-1, IL-1, IL-10, and IL-12p70. While the AGT601S(pOmpA) strain was most potent in this stimulation, DCs were able to produce cytokines in response to the ompA mutant, suggesting that OmpA plays an important role in DC activation but that it is not the sole factor. The use of bacterial mutants that are deficient in the expression of other surface molecules in combination with having the OmpA deficiency would be helpful to address this issue.
Second, it was surprising to find that pretreatment of AGT601S(pOmpA) bacteria with OmpA-specific MAb (IgG2a) or antisera significantly stimulated DCs to produce IL-1, IL-1, and IL-10. Since antibody alone did not have any effect, we speculated that the bacterium-Ab immune complex was responsible for this enhancement in cytokine production. It is well known that human and murine DCs express different types of receptors involved in antigen uptake, such as lectin-type receptors (mannose receptor, DEC205), TLRs, complement receptors, and Fc receptors. The Fc receptor binds to IgG, leading to either DC maturation and activation or DC suppression, depending upon whether the -chain bears an immunoreceptor tyrosine-based activation or an inhibition motif (36). Most studies concur that Abs bind to pathogens or their products to form immune complexes, which facilitate phagocytosis of the pathogen by DCs (18, 52) and presentation of epitopes to major histocompatibility complex class I-restricted CD8+ T cells (2). This Fc receptor-mediated uptake pathogen in DCs is critical for regulating T-cell immunity against intracellular bacterial pathogens, including Listeria monocytogenes (18), Salmonella spp. (45), and Chlamydia spp. (20). This study extends these findings by showing that Ab-bacterium complexes stimulate DCs to produce proinflammatory cytokines, and, more importantly, it emphasizes the role of OmpA in host immune responses to EHEC bacteria.
Since EHEC O157:H7 is an extracellular pathogen in the gut, it was important to examine how the bacteria and their OmpA protein participate in the activation of immune cells in the gut. In mucosal tissues, DCs are scattered throughout intra- and subepithelial sites and the phenotype of these cells is influenced by cytokines and anti-inflammatory mediators that are produced by epithelial cells either under physiological conditions or in response to microbial signals (27). For example, intestinal epithelia up-regulate the expression of the chemokine CCL20 in response to microbial products (such as S. enterica serovar Typhimurium flagellin) or proinflammatory cytokines (9, 38). Up-regulation of CCL20 has been reported to occur during the recruitment of immature DCs in the intestine, especially under inflammatory conditions (12). In an attempt to examine the role of EHEC strains and the OmpA protein in DC activation in a biologically relevant setting, we used an in vitro coculture system. This model system consisted of human colonic Caco-2 cells which were differentiated into apical and basolateral sides and cocultured with human blood-derived DCs in the presence or absence of the wild-type or the ompA mutant strain. Our results indicated that EHEC O157:H7 stimulated DC transmigration through polarized intestinal cells (Fig. 6B) and that this migration occurred in response to secretion of proinflammatory cytokines, especially IL-8, MCP-1, and MIP-1, by Caco-2 cells (Table 3).
This study represents the first report linking the OmpA protein of an enteric pathogen with functions associated with DC activation and cytokine production. Additional experiments are necessary to demonstrate whether EHEC OmpA shares functions similar to those described for K. pneumoniae OmpA during activation of an adaptive immune response or whether differences in DC activation caused by OmpA-positive E. coli strains are due to a different regulatory mechanism controlling their expression or differences on the surface display of this protein.
ACKNOWLEDGMENTS
We thank Joanna Taormina and J. Gerardo Garcia-Gallegos for technical assistance and Mardelle Susman for critical reading of the manuscript.
This work was supported in part by institutional funds from the UTMB John Sealy Memorial Endowment Fund for Biomedical Research and Public Health Service grant DK 56338, which funds the Texas Gulf Coast Digestive Diseases Center, to A.G.T.
Present address: Department of Infectious Diseases, The First Clinical Medical College of Harbin Medical University, Heilongjiang Province 150001, People's Republic of China.
A.G.T. and Y.L. contributed equally to this work.
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Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555-1070
ABSTRACT
Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.
INTRODUCTION
Enterohemorrhagic Escherichia coli (EHEC) strains are a class of pathogenic microorganisms responsible for numerous food- and waterborne outbreaks and can cause illness ranging from nonbloody diarrhea to copious bloody discharge in humans. In some individuals, the disease progresses to a more serious stage, and about 2 to 7% of the diarrheal cases (particularly in the pediatric population) can be fatal due to acute kidney failure (hemolytic-uremic syndrome [HUS]) (reviewed in references 26 and 28). Strains of EHEC O157:H7, the most common EHEC serotype in North America, colonize the intestine and produce multiple determinants which cause the pathology associated with the disease, with Shiga toxin (Stx) being a key feature of virulence.
Stx produced by EHEC strains are responsible for severe damage to epithelial and endothelial cells in the intestinal tract (26, 28) and in the kidneys (reviewed in reference 25), but the sequential events leading to HUS are largely unknown. It has been proposed that Stx-mediated activation of immune cells in the gut initiates the pathology of HUS in the kidneys (11); however, it is unclear whether this process could occur alone or in synergy with other bacterial components. While EHEC O157:H7 can produce a distinct lesion known as the attaching and effacing lesion (24, 49, 51), several proteins, including Iha, Cah, ToxB, OmpA (outer membrane protein A), and the long polar fimbriae, have been identified as putative adhesion factors, facilitating attachment of EHEC to host intestinal epithelial cells. The precise roles of these other factors in disease pathogenesis have not yet been defined (46, 47, 50).
OmpA is one of the major proteins in the membrane of E. coli and is highly conserved among gram-negative bacteria (7). It plays a multifunctional role in the biology of the bacteria, acting as a phage and colicin receptor, serving as a mediator in F-factor-dependent conjugation (35), and maintaining structural integrity of membranes and generation of normal cell shape (40). In E. coli K1 strains, OmpA is also known to be associated with the pathogenesis of neonatal meningitis because it mediates invasion of E. coli K1 to brain microvascular endothelial cells (31) and plays a key role during the initial processes of bacterial adhesion and invasion (30, 31, 37).
More recently, the role of OmpA in adherence was established during the screening of an EHEC O157:H7 transposon insertion mutant library for hyperadherent mutants (48). A mutation of the tcdA gene, encoding a transcriptional activator associated with L-threonine transport and degradation, resulted in elevated expression of OmpA and hyperadherence of EHEC O157:H7 to HeLa and Caco-2 cells (48). Inactivation of ompA in the tdcA mutant abolished the hyperadherent phenotype, and mutation of ompA alone in the wild-type EHEC O157:H7 strain reduced adherence by 13.5%. Furthermore, OmpA-specific antiserum inhibited the adherence of other EHEC O157:H7 strains to HeLa cells. These studies collectively imply an important role for OmpA in mediating adhesion of EHEC O157:H7 strains to host cells (48).
It has been shown that antigen-presenting cells (APCs) can recognize Klebsiella pneumoniae OmpA and are activated by this interaction (16, 41). K. pneumoniae OmpA induces the expression of costimulatory molecules and CD83 on human dendritic cells (DCs) and triggers cytokine production by murine and human macrophages (13, 15). OmpA appears to possess a new type of pathogen-associated molecular pattern (PAMP) because it is conserved among the Enterobacteriaceae family, is essential for bacterial survival and virulence, and can activate APCs (14, 29).
DCs are potent APCs with the unique ability to activate nave T cells (reviewed in reference 23). DCs are widely distributed in the skin and the mucosal tissues in the intestinal tract and airways. Upon recognizing PAMPs via specialized receptors such as the C-type lectin, Fc receptors, and Toll-like receptors (TLRs), DCs detect the presence of bacteria in the host tissues, capturing them or their products, migrate to the T-cell areas in lymph nodes, and activate nave T cells (reviewed in reference 23). During these processes, DCs become mature and are activated to produce diverse cytokines. Over the past few years, much has been learned about the biology of EHEC O157:H7 strains, but it is not known whether these bacteria and their products interact with DCs. It is possible that OmpA of EHEC O157:H7 can function as a PAMP to stimulate DC cytokine production and migration. To test this hypothesis, we generated recombinant OmpA proteins, as well as EHEC O157:H7 strains that differ in expression level of OmpA. Using both murine and human DC systems, we demonstrated in this study that purified OmpA proteins and bacteria expressing high levels of OmpA were efficient stimuli for DCs to produce cytokines and that DC cytokine production was significantly enhanced by the bacterium-antibody (Ab) immune complex. In addition, we provided evidence for bacterially induced migration of DCs through polarized colonic epithelial cells.
MATERIALS AND METHODS
Bacterial strains, plasmids, and reagents. Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were routinely grown in Luria-Bertani (LB) broth or on L agar at 37°C overnight (21). When indicated, the strains were grown in Dulbecco's modified Eagle's medium (Cell Growth/Mediatech, Inc.). Antibiotics were added to LB media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 30 μg/ml; streptomycin, 100 μg/ml; and nalidixic acid, 15 μg/ml. Lipopolysaccharide (LPS) (Salmonella enterica serovar Typhimurium) was purchased from Sigma (St. Louis, MO).
Recombinant DNA techniques and plasmid construction. Plasmid DNA was isolated from overnight bacterial cultures by using a QIAGEN QIAprep plasmid preparation kit and introduced by transformation of RbCl2-competent E. coli DH5 strains or by electroporation of EHEC O157:H7 as described by Dower et al. (8). Standard molecular methods were used for restriction endonuclease analyses, ligation, and construction of recombinant plasmids (21). The ompA gene was amplified from EHEC O157:H7 strain EDL933 by PCR using the primer pair 5'-CCGATATCGGTAGAGTTAATATTGA-3' and 5'-CCTCTAGAAAGCGGTTGGAAATGGAAG-3' (the underlining indicates EcoRV and XbaI sites). The amplicon was digested with EcoRV and XbaI and cloned in the corresponding restriction sites of pACYC184 to create pOmpA.
Excision of the cat cassette from the EHEC 86-24 ompA mutant strain AGT601. Strain AGT601 (48) was created previously by inactivation of the ompA gene with a cat cassette (6). To create the AGT601S strain for this study, the cat gene was excised by FLP recombination, using the temperature-sensitive replication plasmid pCP20, following the protocol previously described (6).
Western blot analysis. Overnight bacterial cultures were adjusted into portions of 2.0 x 108 bacteria, washed in phosphate-buffered saline (pH 7.4), resuspended in 200 μl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer, and boiled for 10 min. Solubilized proteins were separated on 12% SDS-PAGE gels according to the method of Laemmli (19), and proteins were transferred to Immobilon-P membranes (Millipore) by using a Trans-Blot SD transfer cell (Bio-Rad). After nonspecific sites were blocked, the membrane was incubated with rabbit anti-OmpA antibody (1:30,000) and then with goat anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (1:30,000; Sigma) and developed. The OmpA antibodies used in this study have been purified and their specificity previously reported (31).
Expression and purification of the His-tagged OmpA protein. DNA fragments containing the complete open reading frame of ompA from EHEC O157:H7 strain EDL933 were obtained using PCR and the following primers: 5'-CCGAATTCATGAAAAAGACAGCTATCGCGA-3' and 5'-AACTAAGCTTTGCGGCTGAGTTACAACGTC-3' (underlining indicates EcoRI and HindIII sites). The amplicons were cloned into pBad/MycHis (Invitrogen), and the resulting plasmid (pBadOmpA) was introduced into E. coli TOP10 cells. Expression of the gene was induced by the addition of arabinose to a final concentration of 0.2% and incubation at 37°C for 4 h. The His-tagged OmpA protein was purified following protocols recommended by the manufacturers of the ProBond purification system (Invitrogen). The amount of purified protein was quantified by a standard Bradford assay (Bio-Rad) (3a). A Limulus amebocyte lysate test indicated that the different stocks of purified OmpA samples contained 0.86 to 1.6 μg/ml of LPS. To obtain endotoxin-free His-tagged OmpA protein, the purified protein was passed through a Detoxigel Affinity Pak prepacked column (Pierce). However, the yield of OmpA after the elution was only 1/10 of the original concentration (data not shown). Therefore, the experiments were performed in the presence of polymyxin B, as described below.
Generation of BM-DCs. C57BL/6 and C3H/HeJ mice were purchased from Jackson Laboratory (Bar Harbor, ME). A spontaneous mutation in Toll-like receptor 4 (TLR4) occurring at the LPS response locus makes C3H/HeJ mice endotoxin resistant. All mice were maintained under specific-pathogen-free conditions and used for experiments at 6 to 10 weeks of age with protocols approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX). Bone marrow-derived DCs (BM-DCs) were generated as previously described (33), with certain modifications. Briefly, bone marrow cells were cultured in petri dish plates (Fisher Scientific, Houston, TX) at 2 x 106 cells per 10 ml of 10% fetal bovine serum-supplemented Iscove's modified Dulbecco's medium (Invitrogen/Gibco). Culture supernatants of J558L cells that had been transfected with the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene were used as the source of GM-CSF. Nonadherent cells were harvested at day 8 and further cultured in a six-well plate overnight. Resultant nonadherent cells were typically >80% CD11c+ cells as judged by flow cytometric analysis. Sometimes, CD11c+ cells were directly purified from a day-8 culture to >95% purity with microbeads according to the manufacturer's protocol (Miltenyi Biotec, Auburn, CA).
Stimulation of murine DCs. Harvested BM-DCs were washed, suspended in complete Iscove's modified Dulbecco's medium, and stimulated with different concentrations of LPS (0.04, 0.08, or 0.16 μg/ml), purified His-tagged OmpA (0.1, 0.25, 0.35, 0.5, 0.7, or 1.05 μg/ml), or different strains of bacteria at 1.25 x 105 cells/200 μl/well in 96-well plates or 6.25 x 105 cells/0.5 ml/well in 24-well plates. The stimulation culture was not supplemented with any cytokines, including GM-CSF. For some experiments, bacteria were preincubated with anti-OmpA polyclonal Ab (PAb) or monoclonal Ab (MAb) (kindly provided by N. V. Prasadarao and S. P. Singh [39], respectively) before being added to DC cultures. Supernatants were harvested at 24 h for measuring levels of interleukin-1 (IL-1), IL-1, IL-10, and IL-12p40 (see below). To assay the effect of LPS on DC stimulation, samples of His-tagged OmpA or LPS were preincubated for 10 min with 10 μg/ml polymyxin B sulfate (Sigma) before addition to the cells. Supernatants were harvested at 24 h for measuring levels of IL-10 and IL-12p40.
Purification of human DCs. DCs were purified from healthy-human peripheral blood mononuclear cells by negative selection using the magnetic column separation system (StemCell Technologies, Inc.). Monocytes (5 x 105/ml) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, GM-CSF (100 ng/ml), and IL-4 (50 ng/ml) in 24-well plates. Cytokines were replenished every 3 days. Nonadherent, immature DCs were obtained at 7 days, and only immature CD11a+ CD83– DCs were used in the experiments listed below.
Polarized intestinal epithelial cell model. Caco-2 cells, a human adenocarcinoma cell line (American Type Culture Collection, Bethesda, MD), were seeded on membrane inserts in Costar 12-well tissue culture transwell plates (0.4- or 3-μm filters; Corning, Inc., Corning, NY) until confluent. This cell line was used between passages 5 and 15 and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 110 mg/liter of sodium pyruvate, 100,000 IU/liter of penicillin, and 100 mg/liter of streptomycin (Gibco BRL, Grand Island, NY) at 37°C in 5% CO2. The cells were allowed to grow into a confluent, differentiated, polarized monolayer, establishing microvilli, brush borders, and tight junctions.
DC transmigration assay using polarized Caco-2 cells. Caco-2 cells grown on 3-μm filter inserts were infected on the apical (AP) surface with 1 x 107 CFU/ml of EHEC O157:H7 strain 86-24, AGT601S, or AGT601S transformed with a plasmid that carries the ompA gene [AGT601S(pOmpA)] for 1 h at 37°C and 5% CO2. After the prestimulation time, freshly isolated human DCs (1 x 105) were added to the basolateral (BL) chamber and migration was assessed at 4 h. Basolateral DCs with medium only added to the AP chamber served as a negative control. The AP and BL supernatants were collected for counting in a hemacytometer to determine immune-cell migration. Additionally, AP and BL supernatants from DC migration were analyzed for cytokine secretion.
Bacterial-adhesion assays. Caco-2 cells were seeded with 1 x 105 cells/well and incubated for 48 h at 37°C and 5% CO2 in 24-well plates (Corning), and an adhesion assay was performed as described previously (48). Briefly, cell monolayers were washed twice with phosphate-buffered saline and then infected with 1 x 107 bacteria for 4 h or 5 h. Adherence was evaluated qualitatively by Giemsa staining and quantitatively by plating adherent bacteria on L agar plates with the proper antibiotic. In the DC transmigration assay and following immune-cell counting, adherence of bacteria to the AP surface of the polarized cells was evaluated quantitatively by plating serial dilutions of the bacteria, recovered by Triton X-100 treatment (48), on L agar plates with the appropriate antibiotic and growing overnight at 37°C.
Murine and human cytokine assays. To measure the levels of IL-1, IL-1, IL-10, and IL-12p70 in murine DC cultures, specific enzyme-linked immunosorbent assays (ELISAs) were performed with OptEIA kits (BD Biosciences) as previously described (17). For the experiments with human DCs, supernatants were collected, at 5 h after bacterial infection and immune-cell migration, from the AP and BL surfaces of Caco-2 polarized cells. The amount of human cytokine secretion in the supernatants was quantified by a Bio-Rad BioPlex multiplex cytokine system, and this process was performed at the GI Immunology Core facility (UTMB).
Statistical analysis. Statistical differences during bacterial adhesion to epithelial cells were determined by a paired t test. Similarly, to determine the differences in cytokine production by different stimuli, a paired t test was also used.
RESULTS
EHEC OmpA induced DCs to secrete cytokines in a dose-dependent and TLR4-independent manner. DCs discriminate between different microbial pathogens and induce T-cell responses (32). However, it is not clear to what extent EHEC O157:H7 recognition (whole organism or individual microbial components) could induce different functional states of DCs that favor different T-cell effector phenotypes. We examined whether coculturing DCs with purified OmpA induced maturation and specific cytokines known to be secreted by DCs (32). Due to the difficulty in purifying OmpA without contamination by lipoprotein (or endotoxin [see Materials and Methods]), we used DCs derived from C3H/HeJ mice and examined DC cytokine production in response to purified EHEC OmpA and LPS (a negative control). As shown in Fig. 1A through D, OmpA induced DCs to produce high levels of IL-1, IL-1, IL-10, and IL-12p70 in a dose-dependent fashion, while untreated DCs (control) did not produce measurable levels of cytokines. Since exogenous LPS (even at 0.16 μg/ml) did not stimulate DCs to produce these cytokines, these results suggest that purified OmpA activated DCs via a TLR4-independent mechanism.
To further determine whether DC cytokine production was due to OmpA and not to LPS contamination, we generated DCs from C57BL/6 mice and examined DC cytokine production in response to purified EHEC OmpA and LPS in the presence or absence of polymyxin B. Figure 1E and F show that there was a dose-dependent increase in IL-12p70 and IL-10 production in OmpA-stimulated DCs, even in the presence of polymyxin B. In contrast, addition of polymyxin B to the LPS control samples totally abolished cytokine production. Taken together, our results suggested that DC responses were mediated in part via TLR4-independent mechanisms due to OmpA stimulation and in part via TLR4-dependent mechanisms (due to trace amounts of LPS).
EHEC O157:H7 strains with different expression levels of OmpA were capable of stimulating DCs to produce cytokines. We have previously shown that OmpA acts as an adhesin facilitating binding of EHEC O157:H7 to intestinal epithelial cells (48). However, it was unclear in that study whether the adherent phenotype of the ompA mutant could be rescued in trans by complementation with the ompA gene on a plasmid. To test this possibility and to examine the role of OmpA in bacterium- DC interaction, we transformed EHEC strain AGT601S with a plasmid that carries the ompA gene, which is referred to as AGT601S(pOmpA) in this study. Adhesion assays performed at two different time points indicated that EHEC mutant strains AGT601 (ompA::cat) and AGT601S (ompA mutant) adhered to Caco-2 cells at a level comparable to that of our previous findings with HeLa cells (48) but that they adhered to a lower extent (50% lower) than the wild-type 86-24 strain (Table 2). At 5 h postinfection, there was not a statistical difference between the ompA mutants and the wild-type control, probably due to the expression of other adhesins, such as intimin. In contrast, the adherence of strain AGT601S(pOmpA) at 4 h postinfection was significantly higher than that of the wild-type control (Table 2) (P < 0.05). Western blot analysis further confirmed the expression levels of OmpA protein in the different strains (Fig. 2A). While no OmpA expression was detected in strain AGT601S (Fig. 2A, lane 2), it appeared that that strain AGT601S(pOmpA) (Fig. 2A, lane 3) expressed slightly more OmpA protein than did the wild-type strain (Fig. 2A, lane 1). Collectively, these results confirmed and extended our previous findings (48), indicating a role for OmpA in adherence of EHEC to the host cells.
To examine whether DCs responded to these bacterial strains differently, we incubated BM-DCs of C3H/HeJ mice with increasing concentrations of EHEC strain 86-24, AGT601S, AGT601S (pACYC184), or AGT601S(pOmpA) or the E. coli K-12 strain MG1655 (nonpathogenic, OmpA-positive control strain) and measured cytokine production at 24 h postinfection. As shown in Fig. 3, no secretion of cytokines was detected when DCs were left untreated or incubated with bacteria at a bacterium-to-cell ratio of 100:1. At the 500:1 and 1,000:1 ratios, DCs produced high levels of IL-1, IL-1, IL-10, and IL-12p70. DCs exposed to strain AGT601S(pOmpA) consistently produced significantly higher levels of IL-1, IL-10, and IL-12p70 than did the wild-type controls. The cytokine production induced by the complemented ompA mutant strain was not attributed to the presence of the empty vector pACYC184. Although we observed a marginal reduction in expression of IL-1 and IL-10 with the ompA mutant groups, especially with the low-bacterial-dose groups, this trend was not consistent in all of the independent repeats. Finally, we tested whether any OmpA-positive E. coli strain was capable of stimulating cytokine production when DCs were treated with bacterium-to-cell ratios similar to those used with the EHEC strains. Surprisingly, E. coli K-12 strain MG1655 induced DCs to produce low levels of IL-1, IL-10, and IL-12p70 only at the ratio of 1,000:1. Taken together, these studies suggest that overexpression of OmpA on a plasmid was associated with an increase in DC cytokine secretion and that not all OmpA-positive strains were capable of inducing DCs to respond to bacterial stimulus.
Anti-OmpA Abs significantly enhance DC cytokine secretion. We have previously shown that OmpA-specific antiserum inhibited the adherence of different EHEC O157:H7 strains to HeLa cells by approximately 25% (48). We hypothesized that incubation of bacteria with anti-OmpA sera prior to infection would block interaction of OmpA with DCs and therefore reduce DC cytokine secretion. To test this hypothesis, we focused on the strain AGT601S(pOmpA) and used serial dilutions of rabbit anti-OmpA PAb, mouse anti-OmpA MAb (IgG2a), and an isotype control (IgG2a). Surprisingly, we found that in comparison to results for the groups with untreated bacteria, preincubation of bacteria with OmpA-specific Abs (particularly at a 10:1 or a 1:1 dilution of the Ab) significantly increased the production of IL-1, IL-1, and IL-10 (Fig. 4). Preincubation of bacteria with an isotype control also enhanced the production of IL-1 and IL-1 (but not IL-10), but the levels of induction were significantly lower than those produced by OmpA-specific antisera. To examine whether anti-OmpA Ab alone had direct effects on DC cytokine production, we left cells untreated or treated them with serial dilutions of anti-OmpA MAb alone or MAb-preincubated strain AGT601S(pOmpA). It was evident in Fig. 5 that MAb alone was incapable of stimulating DCs to produce cytokines and that the bacterium-MAb immune complex was a potent stimulus for DCs to produce high levels of IL-1, IL-1, and IL-10.
Polarized Caco-2 cells exposed to EHEC O157:H7 were capable of producing cytokines and triggering DC migration. DC activation and migration are essential processes for the functionality of DCs (23). Having demonstrated a role for EHEC OmpA in DC cytokine secretion, we then tested whether EHEC O157:H7, upon binding to host cells, can stimulate the migration of DCs across intestinal epithelial cells. For these experiments, the AP surface of polarized Caco-2 cells was infected with EHEC strains for 1 h, prior to the addition of DCs in the BL chamber. After an additional 4 h of incubation, the AP and BL cell supernatants were collected and analyzed for adherent bacteria, Caco-2 cell cytokine secretion, and DC migration. As shown in Fig. 6A, at 5 h postinfection, we recovered more bacteria of the wild-type EHEC O157:H7 strain 86-24 from the AP surface of Caco-2 cells than we recovered of the ompA mutant strain (P < 0.05). When the ompA mutant was complemented in trans with the pOmpA plasmid, the levels of adhesion were regained; however, they did not fully restore the wild-type phenotype. These transwell filter results were comparable with those obtained with the Caco-2 monolayers (Table 2), suggesting a role for OmpA in facilitating the binding of bacteria to the apical portion of Caco-2 cells.
To determine whether binding of EHEC strains to the AP and BL surfaces of Caco-2 cells could induce cells to produce cytokines, we collected culture supernatants from both chambers at 5 h postinfection and measured cytokine production. It was evident that the binding of wild-type and ompA mutant strains to the AP surface of Caco-2 cells induced the secretion of several cytokines and chemokines, including proinflammatory cytokines (IL-8, CCL2/monocyte chemoattractant protein [MCP-1], and CCL4/macrophage inflammatory protein 1 [MIP-1]), those that augment neutrophil survival (granulocyte colony-stimulating factor and GM-CSF), those that cause activation of cells from circulation (IL-6), and those that induce production of more cytokines (tumor necrosis factor alpha [TNF-]). Furthermore, cytokine production was more prominent in the BL supernatants (Table 3). Basal levels of cytokine secretion were detected when the polarized Caco-2 cells were incubated with DCs in the absence of bacterial stimuli (Table 3); however, they were much lower than levels for those cells stimulated with bacteria. Furthermore, infection of Caco-2 cells with the different bacterial strains, but in the absence of DCs, confirmed that cytokines detected were produced by the intestinal epithelial cells in response to the bacterial stimuli (data not shown). However, we did not observe major differences in cytokine secretion between the wild-type strain and the ompA mutant, regardless of their ability to bind Caco-2 cells, suggesting the possible involvement of bacterial factors other than OmpA in cytokine induction.
Given the broad profile of cytokine production by Caco-2 cells, we reasoned whether binding of EHEC to Caco-2 cells promoted migration of DCs from the BL side to the AP side. We counted the percentage of DCs recovered from each of the two chambers and showed that binding of the wild-type EHEC strain 86-24, its ompA mutant (AGT601S), or the complemented strain AGT601S(pOmpA) to the AP surface of Caco-2 cells promoted DC migration (Fig. 6B). Surprisingly, strain AGT601S was more efficient than the wild-type strain in promoting DC migration to the AP side (P < 0.01) (Fig. 6B). This phenotype was reversible upon introduction of the pOmpA plasmid (Fig. 6B). These data suggest that in the absence of the OmpA protein, other bacterial surface proteins were involved in stimulating DC migration.
DISCUSSION
EHEC O157:H7 strains are known to be noninvasive organisms, to adhere to the apical surface of colon epithelial cells, and to cause infection that is characterized by acute inflammation of the colonic mucosa (26). EHEC is thought to signal colon epithelial cells to produce proinflammatory signals that can chemoattract and activate leukocytes and activate mucosal inflammation (43, 44). Stx and flagellin are examples of EHEC virulence factors that have been reported to stimulate the production of proinflammatory chemokines, resulting in an influx of neutrophils and other leukocytes in cultured human colon cell lines (3, 34, 43, 44). Further, we have recently demonstrated that the major functional signaling pathway for the up-regulated production of proinflammatory chemokines by EHEC-infected human colon epithelium in vivo involves H7 flagellin signaling through TLR5, where Stx signaling is not required (22). No additional data as to whether any other EHEC factor(s) is associated with the recruitment and/or activation of DCs, potent APCs that play a crucial role for host defense against invading pathogens (27), are yet available. We reported in this study that the OmpA protein from EHEC O157:H7 stimulated DC maturation and secretion of cytokines, which is not observed with the nonpathogenic E. coli OmpA-positive strain MG1655. Binding of OmpA-expressing EHEC O157:H7 to the apical surface of polarized Caco-2 cells is not sufficient to stimulate secretion of proinflammatory cytokines and transmigration of DCs, which suggests that other EHEC O157:H7 surface components in addition to OmpA are involved in stimulating DC migration.
Host defense against bacteria is dependent upon clearance by phagocytes (1). Several reports by Jeannin and colleagues have demonstrated that K. pneumoniae OmpA up-regulated the secretion of cytokines (e.g., TNF-, IL-1, IL-10, and IL-12) from murine and human macrophages and bone marrow-derived or purified DCs (4, 15). These in vitro data collectively suggest that OmpA, by up-regulating cytokine production, may favor in vivo leukocyte recruitment to the site of infection and leukocyte activation. In support of this hypothesis, it has recently been demonstrated that subcutaneous injection of K. pneumoniae OmpA in mice triggered a local and transient inflammation accompanied by an increase in draining lymph node cell numbers, with a higher proportion of DCs and macrophages (15). Macrophages are recruited to kill bacteria, and the interaction between OmpA and macrophages initiates a pathway by which innate cells respond to and eliminate pathogens. More-complex cellular and humoral responses to K. pneumoniae OmpA have been suggested by the findings that K. pneumoniae OmpA is recognized by cellular receptors such as LOX-1 and SREC-1 for bacterial binding and is recognized by TLR2 for signaling (13). Therefore, the sequential events include the initial interaction between cellular receptors and K. pneumoniae OmpA, the activation of the proinflammatory signal pathway (including the elevation of humoral PTX3), and the enhanced binding of PTX3 to K. pneumoniae OmpA. Cellular and humoral (PTX3-mediated) recognition are complementary in mediating the innate response to K. pneumoniae OmpA (13).
We have shown in this study that recombinant OmpA of EHEC stimulated murine DCs to produce IL-1, IL-1, IL-10, and IL-12p70 in a dose-dependent and TLR4-independent manner (Fig. 1). At this stage, the receptor(s) that mediates these cytokine responses is unclear. In an attempt to understand the relative contribution of OmpA in DC responses to EHEC bacteria, we examined the DC cytokine responses to EHEC O157:H7 strain 86-24, its isogenic ompA mutant (AGT601S), and the complemented strain AGT601S(pOmpA) and obtained two interesting findings. First, murine DCs responded to bacterial stimulation by producing high levels of IL-1, IL-1, IL-10, and IL-12p70. While the AGT601S(pOmpA) strain was most potent in this stimulation, DCs were able to produce cytokines in response to the ompA mutant, suggesting that OmpA plays an important role in DC activation but that it is not the sole factor. The use of bacterial mutants that are deficient in the expression of other surface molecules in combination with having the OmpA deficiency would be helpful to address this issue.
Second, it was surprising to find that pretreatment of AGT601S(pOmpA) bacteria with OmpA-specific MAb (IgG2a) or antisera significantly stimulated DCs to produce IL-1, IL-1, and IL-10. Since antibody alone did not have any effect, we speculated that the bacterium-Ab immune complex was responsible for this enhancement in cytokine production. It is well known that human and murine DCs express different types of receptors involved in antigen uptake, such as lectin-type receptors (mannose receptor, DEC205), TLRs, complement receptors, and Fc receptors. The Fc receptor binds to IgG, leading to either DC maturation and activation or DC suppression, depending upon whether the -chain bears an immunoreceptor tyrosine-based activation or an inhibition motif (36). Most studies concur that Abs bind to pathogens or their products to form immune complexes, which facilitate phagocytosis of the pathogen by DCs (18, 52) and presentation of epitopes to major histocompatibility complex class I-restricted CD8+ T cells (2). This Fc receptor-mediated uptake pathogen in DCs is critical for regulating T-cell immunity against intracellular bacterial pathogens, including Listeria monocytogenes (18), Salmonella spp. (45), and Chlamydia spp. (20). This study extends these findings by showing that Ab-bacterium complexes stimulate DCs to produce proinflammatory cytokines, and, more importantly, it emphasizes the role of OmpA in host immune responses to EHEC bacteria.
Since EHEC O157:H7 is an extracellular pathogen in the gut, it was important to examine how the bacteria and their OmpA protein participate in the activation of immune cells in the gut. In mucosal tissues, DCs are scattered throughout intra- and subepithelial sites and the phenotype of these cells is influenced by cytokines and anti-inflammatory mediators that are produced by epithelial cells either under physiological conditions or in response to microbial signals (27). For example, intestinal epithelia up-regulate the expression of the chemokine CCL20 in response to microbial products (such as S. enterica serovar Typhimurium flagellin) or proinflammatory cytokines (9, 38). Up-regulation of CCL20 has been reported to occur during the recruitment of immature DCs in the intestine, especially under inflammatory conditions (12). In an attempt to examine the role of EHEC strains and the OmpA protein in DC activation in a biologically relevant setting, we used an in vitro coculture system. This model system consisted of human colonic Caco-2 cells which were differentiated into apical and basolateral sides and cocultured with human blood-derived DCs in the presence or absence of the wild-type or the ompA mutant strain. Our results indicated that EHEC O157:H7 stimulated DC transmigration through polarized intestinal cells (Fig. 6B) and that this migration occurred in response to secretion of proinflammatory cytokines, especially IL-8, MCP-1, and MIP-1, by Caco-2 cells (Table 3).
This study represents the first report linking the OmpA protein of an enteric pathogen with functions associated with DC activation and cytokine production. Additional experiments are necessary to demonstrate whether EHEC OmpA shares functions similar to those described for K. pneumoniae OmpA during activation of an adaptive immune response or whether differences in DC activation caused by OmpA-positive E. coli strains are due to a different regulatory mechanism controlling their expression or differences on the surface display of this protein.
ACKNOWLEDGMENTS
We thank Joanna Taormina and J. Gerardo Garcia-Gallegos for technical assistance and Mardelle Susman for critical reading of the manuscript.
This work was supported in part by institutional funds from the UTMB John Sealy Memorial Endowment Fund for Biomedical Research and Public Health Service grant DK 56338, which funds the Texas Gulf Coast Digestive Diseases Center, to A.G.T.
Present address: Department of Infectious Diseases, The First Clinical Medical College of Harbin Medical University, Heilongjiang Province 150001, People's Republic of China.
A.G.T. and Y.L. contributed equally to this work.
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