Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0904516 on May 13, 2005

Published online before print May 13, 2005
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(Journal of Leukocyte Biology. 2005;78:544-554.)
© 2005 by Society for Leukocyte Biology

Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-{kappa}B activation

Suresh K. Selvaraj* and Nemani V. Prasadarao*,{dagger},1

* Division of Infectious Diseases, The Saban Research Institute, Childrens Hospital Los Angeles, and
{dagger} Keck School of Medicine, University of Southern California, Los Angeles

1 Correspondence: Division of Infectious Diseases, MS #51, The Saban Research Institute, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. E-mail: pnemani{at}chla.usc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1ß, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-{alpha}, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1ß, IL-1ß, and IL-8). In contrast, infection of monocytes with a mutant E. coli, which lacks outer membrane protein A (OmpA– E. coli) resulted in robust production of cytokines and chemokines. Wild-type E. coli K1 (OmpA+ E. coli) prevented the phosphorylation and its degradation of inhibitor of {kappa}B, thereby blocking the translocation of nuclear factor (NF)-{kappa}B to the nucleus. OmpA+ E. coli-infected cells, subsequently subjected to lipopolysaccharide challenge, were crippled severely in their ability to activate NF-{kappa}B to induce cytokine/chemokine production. Selective inhibitors of the extracellular signal-regulated kinase (ERK) 1/2 pathway and p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase, significantly reduced the activation of NF-{kappa}B and the production of cytokines and chemokines induced by OmpA– E. coli, indicating a role for these kinases in the NF-{kappa}B/cytokine pathway. It is interesting that the phosphorylation of ERK 1/2 and p38 MAPK was notably reduced in monocytes infected with OmpA+ E. coli when compared with monocytes infected with OmpA– E. coli, suggesting that the modulation of upstream events common for NF-{kappa}B and MAPKs by the bacterium is possible. The ability of OmpA+ E. coli K1 to inhibit the macrophage response temporarily may enable bacterial survival and growth within the host for the onset of meningitis by E. coli K1.

Key Words: meningitis • OmpA • phagocytosis • inflammation • MAP kinases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial meningitis is the most serious infection of the central nervous system (CNS), resulting in significant neurological sequelae in half of the survivors, such as hearing loss, cortical blindness, and mental retardation [1 2 3 ]. Escherichia coli K1 is the most common gram-negative bacterium associated with neonatal meningitis. For the last 15–20 years, emphasis has been on a greater understanding of the pathogenesis and pathophysiology of this disease in an attempt to improve the outcome; however, the mortality and morbidity rates remain high. Increased systemic concentrations of proinflammatory cytokines have been correlated with septic shock and mortality [4 , 5 ]. However, the interaction of E. coli K1 with phagocytic cells and its role in the production of proinflammatory cytokines have not been studied in detail.

As most cases of meningitis occur via hematogenous spread, and a certain threshold level of bacteremia is required for the onset of the disease, the bacteria must survive in the blood for quite some time. Therefore, neutrophils, monocytes, and macrophages might be the primary cells in the production of cytokines as a result of the interaction with E. coli K1. Our recent studies in the newborn rat model of hematogenous meningitis have demonstrated that E. coli K1 enters monocytes and macrophages and multiplies over a period of time [6 ]. Similar phenomenon was also observed in monocytic and macrophage cell lines. Survival of E. coli K1 in these cells may represent an important step in the vicious cycle of this pathogen for the establishment of a replication-permissive niche within monocytes and/or macrophages. Therefore, E. coli K1 may have developed some unique mechanisms for intracellular survival and development of infection. One such survival strategy used by E. coli K1 in phagocytes is the induction of antiapoptotic protein BclXL expression during the entry, as we reported recently [7 ].

The presence of cytokines, such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1ß, and IL-6 in cerebrospinal fluid (CSF), and blood is a hallmark of the pathogenesis of neonatal meningitis [5 ]. Although it is well-established that endothelial cells, astrocytes, and microglia produce all the cytokines in the CNS, the role of phagocytes in the inflammatory response to E. coli K1 is not known. The major inflammatory stimuli are lipopolysaccharide (LPS) and peptidoglycan for gram-negative and gram-positive organisms, respectively. The interaction of these molecules with phagocyte receptors activates inhibitor of {kappa}B (I{kappa}B) kinase (IKK)-dependent nuclear factor (NF)-{kappa}B pathways and three major mitogen-activated protein kinase (MAPK) pathways, i.e., extracellular signal-regulated kinases 1 and 2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK. Several pathogens, however, have the ability to interfere with the activation of NF-{kappa}B by manipulating a variety of signaling pathways upstream of NF-{kappa}B [8 ].

NF-{kappa}B comprises of a family of closely related transcription factors, which play a key role in the expression of genes involved in inflammation and immune response [9 ]. NF-{kappa}B is a dimeric transcription factor composed of homo- or heterodimers of the Rel family of proteins, of which p50/p105, p52/p100, p65 (RelA), RelB, and c-Rel have been identified in mammalian cells [9 ]. Among these, p50/p65 is the most frequent dimer and hence, considered the prototype. NF-{kappa}B is usually present in an inactive form in the cytoplasm bound to a member of the I{kappa}B family, generally I{kappa}B-{alpha}. In this complex, I{kappa}B blocks the nuclear translocation signal of NF-{kappa}B, and the NF-{kappa}B-I{kappa}B complex needs to be disrupted for NF-{kappa}B to become activated [10 ]. Specific phosphorylation of serine 32 and 36 on I{kappa}B-{alpha} by IKK triggers the disruption of the NF-{kappa}B-I{kappa}B complex, which renders I{kappa}B for ubiquitination and subsequent degradation by the proteosomal complex, thereby releasing NF-{kappa}B from I{kappa}B and allowing nuclear translocation [11 12 13 ]. Once in the nucleus, NF-{kappa}B specifically binds to {kappa}B sites present in promoters and enhancers of multiple genes, resulting in the expression of various proteins, including cytokines, chemokines, cell adhesion molecules, and antiapoptotic proteins [14 ].

In contrast to the general perception that E. coli K1 interaction with all phagocytic cell types results in the production of proinflammatory cytokines and chemokines, here, we demonstrated that E. coli K1 infection of monocytes blocked the production of cytokines and chemokines, even after stimulation by exogenous LPS via inhibition of nuclear translocation of NF-{kappa}B. It is of particular interest that only live E. coli K1 but not heat-killed was able to suppress the production of cytokines from monocytes. Infection of monocytes with wild-type E. coli K1 induced the down-regulation of MAPKs, especially p38 MAPK, when compared with the cells infected with OmpA– E. coli, indicating that wild-type E. coli K1 might modulate the signaling pathways common to MAPKs and NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains
A rifampin-resistant mutant of E. coli K1 strain RS 218 (serotype O18:K1:H7) E44 (designated as OmpA+ E. coli), which invades human brain microvascular endothelial cells in vitro and in vivo, was originally isolated from the CSF of a neonate with meningitis [15 ]. A mutant lacking the entire ompA gene (E91) was generated from strain E44 in two steps. First, a tetracycline-resistance marker was mobilized from DME 558, which contains the marker neo-ompA, by P1 transduction to E. coli K12 strain BRE51 in which the ompA gene had been deleted [16 ]. Then, a P1 lysate was used to transduce E. coli K1 strain E44. Tetracycline-resistant colonies were screened for lack of OmpA expression by Western blot analysis to identify mutant E91. OmpA– E. coli (E91) expresses all other surface structures including LPS, K1 capsular polysaccharide, and S-fimbriae. E91 was complemented with a plasmid containing ompA (designated as pOmpA+ E. coli) and expresses similar levels of OmpA to that of E44 [15 ]. E91 strain was also transformed with the plasmid alone to obtain pOmpA– E. coli. Sfa– (S-fimbriae), IbeA–, and IbeB– E. coli strains were generated by TnPhoA mutagenesis of E44 as described previously [17 , 18 ]. Heat-killed bacteria were prepared by exposing OmpA+ and OmpA– E. coli to 80°C in a heating block for 30 min. The bacteria were grown in a brain-heart infusion broth with appropriate antibiotics as necessary. All bacterial media were purchased from Difco Laboratories (Detroit, MI).

Reagents, antibodies, and cells
LPS isolated from E. coli 026:B6 was obtained from Sigma Chemical Co. (St. Louis, MO). Antibodies to p50, p65, I{kappa}B, and ß-actin were obtained from Santa Cruz Biotechnology (CA). Antibodies to phosphorylated and nonphosphorylated forms of I{kappa}B, ERK 1/2, p38, and JNK were obtained from Cell Signaling (Beverly, MA). Antibody to histone core was purchased from Maine Biotechnology Services (Portland). PD98059, SB203580, SP600125, and caffeic acid phenetyl ester (CAPE) were obtained from Calbiochem (San Diego, CA). THP-1, a promonocytic cell line, was purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum to a density of 1 x 106 cells/ml and subcultured periodically.

Isolation of peripheral blood monocytes (PBMs)
Human blood from healthy adults was collected using standard sterile conditions with the approval of the Committee on Clinical Investigation at Childrens Hospital Los Angeles (CA). PBMs were isolated using Nycoprep, according to the manufacturer’s instructions. Briefly, 10 vol blood sample (30 ml) was mixed with 1 vol (3 ml) of a solution composed of 6% dextran 500 in 0.9% NaCl. The tube was allowed to stand at room temperature for 45 min, which resulted in the sedimentation of erythrocytes. The leukocyte-rich plasma was harvested, layered over Nycoprep media, density 1.068 g/ml (Accurate Chemical and Scientific, Westbury, NY), at a ratio of 2:1, and centrifuged at 600 g for 15 min. Finally, these cells were allowed to adhere for 1–2 h in a 60-mm Petri dish in a tissue-culture incubator. By this procedure, nonadherent lymphocytes were removed, and the PBMs were used for the experiments.

RNase protection assay (RPA)
THP-1 monocytes were treated with OmpA+ E. coli (1:1 bacteria/cell ratio) and different mutants for 1–4 h, and total RNA was isolated from cultured cells with TriZOL reagent (Life Technologies, Gaithersburg, MD). Assays were performed with a custom-made Riboquant multi-probe RPA system (PharMingen, San Diego, CA). In brief, the isolated RNA (10 µg) was hybridized with 32P-labeled probes overnight at 56°C followed by RNase digestion, according to the manufacturer’s instructions. After digestion, the protected fragments were resolved on a 5% denaturing polyacrylamide gel and transferred to Whatman filter paper No. 3, which was dried and later exposed to X-ray film. The intensity of bands corresponding to TNF-{alpha}, macrophage-inflammatory protein-1ß (MIP-1ß), IL-1ß, IL-8, regulated on activation, normal T expressed and secreted (RANTES), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was analyzed using ImageJ software (http://rsb.info.nih.gov/ij). Values are expressed as relative expression of mRNA normalized to housekeeping GAPDH mRNA.

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
THP-1 cells were treated with different bacteria for various periods, and nuclear extracts were prepared as described previously [19 ]. Briefly, the cells were washed with cold phosphate-buffered saline (PBS) and were resuspended in 400 µl cell lysis buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF)]. The cells were allowed to swell on ice for 10 min, after which, 20 µl 10% Nonidet P-40 (NP-40) was added and mixed thoroughly, followed by centrifugation at 3000 g for 1 min. The supernatant was stored as cytoplasmic extract, and the pellet was suspended in 100 µl buffer C [20 mM Hepes (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF], incubated on ice for 30 min, and vortexed at frequent intervals. The nuclear extract was obtained by centrifuging at 14,000 g for 10 min at 4°C and stored at –70°C until further use.

Nuclear extracts from cells treated as described earlier were analyzed by EMSA, essentially as described previously [20 ]. In brief, the oligonucleotides containing the consensus binding sites for NF-{kappa}B (Santa Cruz Biotechnology) were radiolabeled with 32P using {gamma}32P-adenosine 5'-triphosphate (ATP) and T4 polynucleotide kinase (Invitrogen, Carlsbad, CA) and purified using NucTrap probe purification columns (Stratagene, La Jolla, CA) according to the instruction manual. DNA-binding reaction was performed in a 15-µl mixture containing 3–4 µg nuclear extract, 5 µl 3x binding buffer (60 mM HEPES, pH 7.9, 3 mM DTT, 3 mM EDTA, 150 mM KCl, and 12% Ficoll), 1.0 µg poly(dI-dC; Roche Diagnostics, Indianapolis, IN), and 20,000 counts per minute radiolabeled probe. To demonstrate the specificity of the DNA-protein interaction, a 50-fold excess of unlabeled, double-stranded oligonucleotide probe was added 10 min before the addition of the labeled probe. In supershift assays, nuclear extracts were incubated with radiolabeled probe followed by the addition of 2 µg antibody specific to each transcription factor. After 25 min incubation on ice, resultant nucleoprotein complexes were resolved from unbound DNA on a native 5% polyacrylamide gel and were detected by autoradiography of the dried gel.

Cytokine and chemokine measurements
THP-1 cells (1x106/ml) were incubated in RPMI containing 5% serum in the presence of the bacteria at a multiplicity of infection (bacteria-to-cell ratio) of 1:1 for various periods. At the end of the indicated periods, ranging from 1 to 8 h, the medium was collected, and cell debris was removed by low-speed centrifugation at 5000 g for 10 min. Supernatant was stored at –80°C until further use. Levels of TNF-{alpha}, IL-1ß, MIP-1ß, RANTES, and IL-8 in the clarified supernatants were assayed using a specific DuoSet enzyme-linked immunosorbent assay (ELISA) development system (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Western blotting
THP-1 cells were treated with OmpA+ and OmpA– E. coli for the respective time periods and then washed with ice-cold RPMI four times to remove any loosely adherent bacteria. The cells were then lysed using radio immunoprecipitation assay buffer [PBS containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 g/ml PMSF, 2 mmol/L aprotinin, and 1 mmol/L sodium orthovanadate]. The cells were disrupted mechanically by sonication and followed by centrifugation at 5000 g to remove cell debris. The protein content in the supernatant was estimated, and 30 µg cell lysate was separated on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane. The membrane was blocked using PBS-Tween 20 containing 5% milk and then probed with the appropriate antibody. The blot was then stripped, blocked, and reprobed using additional antibodies. The immunoreactive proteins were detected with a chemiluminescence detection kit (Super Signal chemiluminescence kit, Pierce Co., Rockford, IL). The protein bands on the X-ray films were scanned, and the bands were quantitated using ImageJ software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Suppression of cytokines and chemokines in THP-1 cells infected with OmpA+ E. coli
As mentioned earlier, the interaction of wild-type E. coli K1 (designated as OmpA+ E. coli) with monocytes or macrophages has not been studied in detail. Our recent data showed that OmpA+ E. coli not only enters both of these cells but also survives and multiplies within the cells [6 ]. In contrast, OmpA– E. coli was taken up by these cells in considerably lower numbers and was killed between 4 and 8 h post-infection. Thus, we speculated that survival of OmpA+ E. coli within monocytes or macrophages would induce significant production of cytokines and chemokines. To examine the cytokine/chemokine profile after infection with OmpA+ or OmpA– E. coli, mRNA levels of a panel of inflammatory cytokines and chemokines were performed by RPA analysis. The intensity of each band was determined with a densitometer and graphed after normalizing to the intensity of GAPDH. As shown in Figure 1A and 1B , the mRNA level of TNF-{alpha} in THP-1 cells infected with OmpA– E. coli was 50% greater than that of OmpA+ E. coli-infected cells up to 4 h. The mRNA levels of RANTES and MIP-1ß were 70–80% lower in OmpA+ E. coli-infected cells than those of OmpA– E. coli-infected cells. It is interesting that the IL-1ß level was inhibited completely by OmpA+ E. coli infection even at 4 h relative to OmpA– E. coli-induced mRNA levels. The mRNA levels of IL-8 were also inhibited several fold by OmpA+ E. coli compared with OmpA– E. coli at 1 h post-infection, and the expression was increased gradually by 4 h. However, the level of IL-8 mRNA induced by OmpA+ E. coli is 40–50% lower than that of OmpA– E. coli-induced IL-8 expression. THP-1 cells were incubated with purified LPS (100 ng/ml) for 1 h as a positive control in these experiments, which showed significant activation of all the cytokines/chemokines. It is interesting that heat-killed OmpA+ E. coli induced the expression of these cytokines and chemokines in a similar manner to OmpA– E. coli-infected cells (data not shown). Preincubation of THP-1 cells with OmpA+ E. coli for 1 h prior to LPS treatment significantly inhibited the LPS-induced cytokines and chemokine mRNA levels, whereas infection with OmpA– E. coli or heat-killed OmpA+ E. coli did not. These results suggest that OmpA+ E. coli infection of THP-1 cells resulted in the suppression of the gene expression of RANTES, MIP-1ß, IL-1ß, and IL-8 more significantly and of TNF-{alpha} moderately compared with OmpA– E. coli. In addition, bacterial viability is important for this inhibition.



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  		Figure 1. Cytokine mRNA levels in THP-1 cells following infection with OmpA+ and OmpA– E. coli. (A) THP-1 cells were uninfected or infected with OmpA+ or OmpA– E. coli for varying periods; total mRNA was isolated and subjected to RPA analysis for the indicated cytokines and chemokines. In separate experiments, the cells were infected with live or heat-killed (HK) bacteria for 1 h, washed, and further incubated with LPS (100 ng/ml) for 1 h. These experiments were carried out at least three times with similar results. Lane C, Control. (B) The densities of the bands were calculated using ImageJ software after scanning the blots, normalized to the density of GAPDH, and graphed.

 
To confirm that the ability of OmpA+ E. coli to induce the suppression of proinflammatory cytokine and chemokine gene expression was a result of OmpA expression, the OmpA– E. coli was complemented with a plasmid containing the ompA gene. The resulting bacteria, pOmpA+ E. coli, express normal levels of OmpA, whereas OmpA– E. coli transformed with plasmid alone (pOmpA– E. coli) showed no expression [15 ]. As shown in Figure 2A and 2B , THP-1 cells infected with pOmpA– E. coli for 2 h induced the mRNA levels of TNF-{alpha}, IL-1ß, IL-8, MIP-1, and RANTES in similar levels to that of OmpA– E. coli-infected cells. However, pOmpA+ E. coli showed significant inhibition of cytokine and chemokine expression. In addition, to determine the contribution of other E. coli structures in inducing the cytokine/chemokine gene expression, E. coli mutants that lack IbeA, IbeB, and Sfa were also used to infect THP-1 cells. OmpA, IbeA, and IbeB are shown to play a role in the invasion of human brain microvascular endothelial cells, whereas Sfa plays a role in the binding of bacteria to host cells [15 , 17 , 18 , 21 ]. It is interesting that E. coli, which do not express IbeA but still express OmpA, also induced cytokine and chemokine expression similar to that of OmpA– E. coli. This result is not unexpected, as our previous studies with endothelial cells indicated that the structural integrity of OmpA might depend on the presence of other surface structures on E. coli, especially IbeA [17 ]. Therefore, lack of suppression of cytokine and chemokine expression by Ibe– E. coli could be a result of the inability of OmpA binding to its cognate receptor in monocytes. In contrast, the absence of IbeB and Sfa did not affect the ability of OmpA+ E. coli to suppress the proinflammatory cytokine gene expression. Thus, taken together, these results demonstrate that OmpA expression is critical for the invasion of monocytes by E. coli K1 and for subsequent events leading to the suppression of proinflammatory molecules.



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  		Figure 2. RPA of THP-1 cells infected with different mutants of E. coli K1. (A) THP-1 cells were incubated for 2 h with wild-type (OmpA+), OmpA– E. coli, or OmpA– E. coli complemented with a plasmid containing full-length ompA gene (pOmpA+) or plasmid alone (pOmpA–) or different mutants, which lack IbeA, IbeB, or Sfa in separate experiments. Total mRNA was isolated and subjected to RPA analysis to determine the cytokine/chemokine mRNA levels. (B). The densities of the bands were calculated using ImageJ software, normalized to the density of GAPDH, and graphed.

 
Proinflammatory cytokine production in THP-1 and PBMs infected with OmpA+ and OmpA– E. coli
Next, we determined if the decreased transcription of cytokines/chemokines translated to decreased protein expression. The release of cytokines/chemokines from THP-1 cells infected with OmpA+ or OmpA– E. coli was determined over a period of 8 h. During these experiments, the number of viable cells was determined by counting the cells using a light microscope per field. The number of viable cells was similar between the two bacterial treatments up to 4 h post-infection but was reduced by 30–40% after 8 h with both treatments (data not shown). As shown in Figure 3A , OmpA+ E. coli induced small quantities of cytokines/chemokines at any given period, whereas OmpA– E. coli induced a two- to threefold increase in the production of all cytokine/chemokines in THP-1 cells at 4 and 8 h post-infection (~60 pg/ml for OmpA+ E. coli vs. 150–200 pg/ml for OmpA- E. coli). LPS treatment of these cells for 8 h showed a profound effect on the production of TNF-{alpha} and IL-8 (~650 pg/ml) when compared with RANTES, MIP-1ß, and IL-1ß (Fig. 2B) . However, preinfection of THP-1 cells with OmpA+ E. coli completely prevented the LPS-induced production of TNF-{alpha} and IL-8, whereas OmpA– E. coli infection showed no such dramatic effect. Heat-killed OmpA+ and OmpA– E. coli produced similar levels of cytokines and chemokines to the levels induced by OmpA– E. coli. Infection of THP-1 cells with heat-killed OmpA+ E. coli prior to LPS treatments did not block the production of these cytokines and chemokines.



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  		Figure 3. Inhibition of cytokine and chemokine production by THP-1 and PBMs infected with OmpA+ E. coli. THP-1 cells (A and B) or PBMs (C and D) were uninfected (Control) or infected with OmpA+ or OmpA– E. coli for 1 h, 4 h, and 8 h. In some experiments, the cells were treated with LPS (100 ng/ml) with or without preinfection with live bacteria or heat-killed (HK) bacteria for 8 h (B and D). The supernatants were collected and assayed by ELISA for indicated cytokines and chemokines. The data represent an average of three separate experiments performed in triplicate ± SD.

 
In addition, we examined the production of proinflammatory cytokines in human PBMs infected with E. coli. As shown in Figure 3C , OmpA– E. coli induced significant production of MIP-1ß and IL-8 by 1 h post-infection, which was further increased by 8 h. In contrast, OmpA+ E. coli induced moderate levels of MIP-1ß and IL-8 at 1 h, which were further increased at 4 and 8 h post-infection, although the levels were 50% lower than that of the levels induced by OmpA– E. coli. TNF-{alpha}, RANTES, and IL-1ß production by PBMs infected with OmpA+ E. coli was significantly lower at all time-points. Similar to the cytokine and chemokine production by LPS in THP-1 cells, PBMs also showed more production of TNF-{alpha}, MIP-1ß, and IL-8 after LPS treatment, which was suppressed significantly by preinfecting the cells with OmpA+ E. coli but not with OmpA– E. coli (Fig. 3D) . Heat-killed OmpA+ E. coli could not inhibit the production of these cytokines and chemokines induced by LPS. THP-1 and PBMs, infected with bacteria, were monitored for cytokine and chemokine production up to 24 h; however, no further, significant increase in the production of the cytokines and chemokines was observed (data not shown). Moreover, the difference between the levels of cytokines and chemokines induced by OmpA+ and OmpA– E. coli was almost similar to the difference observed at 8 h. The number of viable cells at 24 h post-infection with either of the bacteria is only 20–30% when compared with the uninfected cells. Therefore, lack of increase in the production of cytokines and chemokines at 24 h post-infection could be a result of lack of viable cells in the incubation mixture. Taken together, these results suggest that THP-1 cells and PBMs behave similarly in proinflammatory cytokine production, although slight variations in the amounts secreted were observed. In addition, infection with live OmpA+ E. coli is required to mediate suppression of cytokine and chemokine production.

OmpA+ E. coli infection induces the suppression of NF-{kappa}B activation in monocytes
The invasion of host by a pathogen is frequently associated with the activation of NF-{kappa}B, which coordinates various aspects of immune function [14 ]. Therefore, to evaluate the role of NF-{kappa}B activation in the THP-1 cells and PBMs in response to E. coli infection, we analyzed the DNA-binding activity of NF-{kappa}B by EMSA using a radioactive-labeled consensus oligonucleotide {kappa}B probe. Cells were infected with OmpA+ or OmpA– E. coli, and at different time-points after challenge, nuclear protein extracts were prepared and assayed for NF-{kappa}B DNA-binding activity. As shown in Figure 4 , infection with OmpA– E. coli for 1–4 h strongly induced the formation of a protein-DNA complex in THP-1 and PBMs, which was blocked by a 50-fold molar excess of unlabeled NF-{kappa}B oligonucleotides. It is interesting that OmpA+ E. coli-infected cells showed no such increase. LPS treatment of THP-1 cells for 1 h triggered pronounced translocation of NF-{kappa}B. Preinfection of THP-1 cells with OmpA+ E. coli for 1 h inhibited the activation of NF-{kappa}B in response to subsequent challenge with LPS for an additional hour. In contrast, preinfection with OmpA– E. coli or heat-killed OmpA+ E. coli or OmpA– E. coli could not suppress the LPS-induced activation of NF-{kappa}B. These results suggest that live OmpA+ E. coli exerted an inhibitory effect on NF-{kappa}B activity, which could not be overcome by subsequent LPS stimulation.



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  		Figure 4. Translocation of NF-{kappa}B to nuclei is inhibited in THP-1 cells and PBMs infected with OmpA+ E. coli. THP-1 cells and PBMs were cultured in medium alone or infected with OmpA+ or OmpA– E. coli for 1, 2, and 4 h. The cells were also incubated with LPS for 1 h with or without preinfection with live bacteria or heat-killed (HK) bacteria. Nuclear extracts were prepared and subjected to EMSA analysis using a radioactive-labeled probe for a NF-{kappa}B consensus sequence. These experiments were repeated three times with similar results. Lane C, Control.

 
NF-{kappa}B complex predominantly contains RelA (p65) and p50 proteins as heterodimers or homodimers [11 ]. Thus, we examined which of these complexes is involved in OmpA– E. coli-induced activation of NF-{kappa}B by incubating the nuclear extracts with anti-p65 and anti-p50 antibodies prior to EMSA. The complexes were supershifted by the addition of anti-p50 or anti-p65 antibody (Fig. 5A ), suggesting that OmpA– E. coli-induced NF-{kappa}B binding activity is composed of p50 and p65 subunits. To further confirm the translocation of the p65/p50 complex to nucleus, total cell lysates of THP-1 cells infected with the bacteria were separated into cytoplasmic and nuclear fractions, which were subjected to immunoblotting with anti-p50 and anti-p65 antibodies. As shown in Figure 5B , the cytoplasmic extract of OmpA+ E. coli-infected cells revealed the presence of equal quantities of p50 at 1 h compared with that of untreated cells, which was reduced by 20–30% at 2 h, as determined by densitometric analysis. In contrast, the intensity of the p50 protein was decreased by 85% at 2 h in OmpA– E. coli-infected cells. The translocation of p50, however, increased in the nuclear fractions in OmpA– E. coli-infected cells significantly when compared with untreated cells, whereas OmpA+ E. coli infection of THP-1 cells induced no such migration of p50 to the nucleus. Similarly, p65 also translocated from cytoplasm to the nucleus in significant quantities in OmpA– E. coli-infected cells as compared with OmpA+ E. coli-infected and untreated cells. The differences in the amounts of p50 and p65 in cytoplasmic and nuclear fractions are not a result of the presence of unequal quantities of proteins, as the blots stained with anti-ß-actin and antihistone antibodies showed equal amounts of these proteins. These results demonstrated that wild-type OmpA+ E. coli suppresses translocation of NF-{kappa}B to the nucleus.



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  		Figure 5. Infection of THP-1 cells with OmpA+ E. coli inhibits the migration of p50/p65 subunits of NF-{kappa}B to nuclei. (A) THP-1 cells were infected with OmpA– E. coli for 2 h, and nuclear extracts were prepared and subjected to supershift analysis using anti-p50 and anti-p65 antibodies (Ab). Unlabeled probe (+Cold probe) was added in the supershift assay to examine the specificity. (B) Total cell lysates of THP-1 cells infected with OmpA+ or OmpA– E. coli for varying times were separated into cytoplasmic and nuclear extracts and subjected to immunoblotting with anti-p50 and anti-p65 antibodies (1:5000 dilution). In addition, the blot containing cytoplasmic extracts was reprobed with anti-ß-actin antibody, and the blot with nuclear extracts was reprobed with antihistone core antibody. Extracts of uninfected cells (lanes C) were used as controls.

 
OmpA+ E. coli interaction with THP-1 cells reduces the phosphorylation of I{kappa}B-{alpha} and its subsequent degradation
The blockade of NF-{kappa}B translocation led us to examine whether phosphorylation-driven I{kappa}B-{alpha} degradation is defective in OmpA+ E. coli-infected cells. Figure 6 shows a representative immunoblot analysis using antibodies specific to phospho-I{kappa}B-{alpha} and for total I{kappa}B-{alpha}. OmpA– E. coli induced I{kappa}B-{alpha} phosphorylation rapidly for up to 2 h, and a peak phosphorylation was between 15 and 30 min post-infection. Phosphorylation of I{kappa}B-{alpha} resulted in an upward shift of the I{kappa}B-{alpha} band in the SDS-PAGE because of slower electrophoretic mobility. In contrast, OmpA+ E. coli infection did not induce significant phosphorylation of I{kappa}B-{alpha} at any time-point. Densitometric analysis of the bands normalized to ß-actin demonstrated that OmpA+ E. coli infection-induced phosphorylation of I{kappa}B-{alpha} is 70% lower than that of the amount induced by OmpA– E. coli (Fig. 6B) . These results imply that infection of THP-1 cells with OmpA+ E. coli inhibits the phosphorylation of I{kappa}B-{alpha} and subsequently, its degradation to inhibit the NF-{kappa}B activation.



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  		Figure 6. Phosphorylation and degradation of I{kappa}B are inhibited in THP-1 cells infected with OmpA+ E. coli. THP-1 cells were infected with OmpA+ or OmpA– E. coli for varying periods, and total cells lysates were prepared and subjected to immunoblotting with antiphospho-I{kappa}B (pI{kappa}B), anti-I{kappa}B, and anti-ß-actin antibodies (1:1000 dilution). The blots were then probed with horseradish peroxidase-conjugated secondary antibody followed by incubation with enhanced chemiluminescence reagent and then exposed to an X-ray film. C, Control. (B) The blots were scanned, and the intensities of the bands were determined by a densitometer. The density of each band was normalized to the intensity of the corresponding ß-actin band and then graphed.

 
Inhibition of proinflammatory cytokine production and NF-{kappa}B activation by inhibitors of NF-{kappa}B, ERK 1/2 pathway, and p38 MAPKs in THP-1 cells infected with OmpA– E. coli
Our studies thus far have demonstrated that OmpA+ E. coli infection inhibited the NF-{kappa}B activation in THP-1 cells, which may be responsible for the down-regulation of proinflammatory cytokine production. To confirm the role of NF-{kappa}B in cytokine and chemokine production and to investigate the possible role of MAPKs, we used specific inhibitors in the infection assays. First, THP-1 cells were pretreated with the inhibitors for 1 h followed by infection with OmpA– E. coli for 2 h, and RPA analysis was performed. As expected, CAPE (10 µM), an inhibitor of NF-{kappa}B, significantly blocked the up-regulation of mRNA levels of all cytokines and chemokines induced by OmpA– E. coli (Fig. 7A and 7B ). In addition, CAPE profoundly inhibited the up-regulation of mRNA levels of all these cytokines and chemokines induced by LPS (data not shown). Treatment of cells with SB203580 (1 µM), an inhibitor of p38 MAPK, markedly diminished the mRNA levels of all cytokines and chemokines induced by OmpA– E. coli. The ERK 1/2-MAPK pathway inhibitor, PD980589 (10 µM), conversely, significantly blocked the expression of RANTES, MIP-1ß, IL-1ß, and IL-8 but moderately affected that of TNF-{alpha}. In contrast, SP600125 (100 nM), an inhibitor of the JNK-mediated pathway, did not alter the mRNA levels of all cytokines/chemokines. Measurement by ELISA of cytokine and chemokine levels induced by OmpA– E. coli in THP-1 cells pretreated with CAPE, PD98059, and SB203580 also showed significant reduction in the production of TNF-{alpha}, MIP-1ß, IL-1ß, IL-8, and RANTES (Fig. 7C) . No effect was observed in cells treated with the JNK pathway inhibitor.



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  		Figure 7. Inhibitors of ERK 1/2 and p38 kinases significantly blocked the activation of cytokine/chemokine expression and NF-{kappa}B activation induced by OmpA– E. coli. (A) THP-1 cells were infected with OmpA– E. coli for 1 h with or without preincubation with various inhibitors; total mRNA was isolated and subjected to RPA assay. (B) The densities of the bands were calculated using ImageJ software, normalized to the density of GAPDH, and graphed. (C) THP-1 cells were preincubated with various inhibitors for 1 h and then infected with OmpA– E. coli for 8 h, and supernatants were collected and analyzed for the presence of various cytokines/chemokines by ELISA. (D) In separate experiments, the cells were treated as mentioned, and nuclear extracts were isolated and subjected to EMSA analysis. (E) THP-1 cells were pretreated with various MAPK inhibitors for 1 h followed by infection with OmpA– E. coli for varying times as indicated, and cell lysates were prepared and subjected to immunoblotting with antibodies to phospho-I{kappa}B (pI{kappa}B), I{kappa}B, and ß-actin. C, Control.

 
Furthermore, the nuclear extracts of THP-1 cells pretreated with MAPK inhibitors followed by bacterial infection for 1 h were subjected to EMSA. The results also demonstrated that ERK 1/2 and p38 MAPK but not JNK play an important role in the activation of NF-{kappa}B (Fig. 7D) . THP-1 cells treated with CAPE showed complete inhibition of NF-{kappa}B activation or translocation. Taken together, these results suggest that NF-{kappa}B activation and production of cytokines and chemokines by OmpA– E. coli may also require the activation of ERK 1/2 and p38 MAPKs rather than JNK. As the MAPK inhibitors inhibited the DNA-binding activity of NF-{kappa}B, we further examined the effect of these inhibitors on the phosphorylation of I{kappa}B. THP-1 cells were infected with OmpA– E. coli for indicated periods of time with or without prior treatment with MAPK inhibitors; total cell lysates were prepared and subjected to Western blotting with antibodies to phospho-I{kappa}B, I{kappa}B, and ß-actin. As we have shown in the previous experiments, OmpA– E. coli induced the phosphorylation of I{kappa}B up to 30 min, which was declined by 60 min post-infection. This increased phosphorylation of I{kappa}B was inhibited significantly in cells treated with the p38 MAPK inhibitor at every time-point, and PD98059 (ERK 1/2 pathway inhibitor) showed slight inhibition up to 30 min and complete inhibition at 60 min post-infection. The JNK inhibitor also exhibited moderate suppression of I{kappa}B phosphorylation. These results suggest that ERK 1/2 and p38 MAPKs might be playing a major role in the signaling events leading to phosphorylation of I{kappa}B.

OmpA+ E. coli infection of THP-1 cells modulates the phosphorylation of MAPKs
As inhibitors of ERK 1/2 pathway and p38 MAPK blocked the OmpA– E. coli-induced activation of NF-{kappa}B and the production of cytokines, we speculated that OmpA+ E. coli might modulate the activation of these MAPKs in THP-1 cells. Therefore, total cell lysates of THP-1 cells infected with OmpA+ or OmpA– E. coli were examined by Western blotting with antiphospho-ERK 1/2, -p38, and -JNK antibodies. The cells infected with OmpA+ E. coli exhibited increased phosphorylation of ERK 1/2 at 1 h when compared with uninfected cells, which was reduced by 50% at 2 h (Fig. 8A ). In contrast, infection of THP-1 cells with OmpA– E. coli incessantly induced the activation of ERK 1/2 for up to 2 h when compared with untreated cells and cells treated with OmpA+ E. coli (Fig. 8B) . Of note, increased phosphorylation of p38 MAPK was observed from 15 min in THP-1 cells infected with OmpA– E. coli. In contrast, OmpA+ E. coli infection of THP-1 cells showed reduced levels of phosphorylation of p38 MAPK at all time-points, which were 50% lower that that of OmpA– E. coli-induced phosphorylation (Fig. 8B) . The phosphorylation of JNK was 50–60% greater in THP-1 cells infected with OmpA– E. coli at 1 h when compared with OmpA+ E. coli; however, decreased to same levels by 2 h in both bacterial treatments.



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  		Figure 8. Inhibition of ERK 1/2 and p38 MAPK activation in THP-1 cells infected with OmpA+ E. coli. THP-1 cells uninfected (lane C) or infected with OmpA+ or OmpA– E. coli for varying periods; total cell lysates were prepared and subjected to immunoblotting with antiphospho-ERK 1/2, -p38, and -JNK (pERK1/2, pp38, and pJNK1/2, respectively) antibodies. The blots were stripped and reprobed with antibodies to ERK 1/2, p38, and JNK. In separate experiments, the cells were also treated with LPS (100 ng/ml) for 1 h, with or without preinfection with live bacteria or heat-killed (HK) bacteria for 1 h. These experiments were repeated at least three times with similar results. (B). The densities of the phosphorylated protein bands were calculated using ImageJ software, normalized to the density of nonphosphorylated proteins, and graphed.

 
LPS, used as a positive control in these experiments, as expected, induced robust phosphorylation of ERK 1/2 by 1 h, which was abolished completely when the cells were preincubated with OmpA+ E. coli for 1 h but not with OmpA– E. coli. In contrast, heat-killed OmpA+ E. coli lost its ability to inhibit LPS-induced activation of ERK 1/2. PD98059 (10 µM) was also used in these experiments to determine the specificity of inhibitor on ERK 1/2, which showed blocking of ERK 1/2 phosphorylation induced by OmpA– E. coli. Infection of THP-1 cells with OmpA+ E. coli prior to the treatment with LPS significantly inhibited the phosphorylation of p38 MAPK and JNK 1/2 induced by LPS. Inhibition of the phosphorylation of p38 MAPK induced by LPS in the presence of SB203580 is unexpected in our studies, as the inhibitor blocks the binding of ATP to p38 MAPK but not the phosphorylation of the kinase [22 , 23 ]. However, similar results were also observed with group B Streptococcus (GBS) infection of monocytes [24 ]. Conversely, the JNK inhibitor did not block the phosphorylation of JNK induced by LPS, as it blocks the downstream signaling events to JNK [25 ]. Taken together, these data indicate that OmpA– E. coli induces the activation of p38 MAPK at early time-points when compared with ERK 1/2 or JNK, whereas OmpA+ E. coli may interfere with the signaling pathways leading to the phosphorylation of MAPKs. This diminished MAPK activation in monocytes infected with E. coli K1 may account, at least in part, for the decreased production of cytokines and chemokines.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
E. coli K1 is the most common gram-negative bacterium that causes meningitis during the neonatal period [1 2 3 ]. A hallmark of this disease is the presence of high levels of cytokines in blood and CSF, which is a result of the stimulation of phagocytic cells by circulating, free LPS shed by the bacteria. The expression of cytokines is often controlled by activation of the transcription factor NF-{kappa}B and is a result of phosphorylation and degradation of inhibitory I{kappa}B proteins. Previous studies from our lab have shown that E. coli K1 enters, survives, and multiplies in monocytes and macrophages, for which OmpA expression is required [6 ]. Thus, we expected that the infection of monocytes and macrophages by E. coli K1 results in the production of significant amounts of cytokines. It is surprising that we observed that E. coli K1 rapidly down-regulates the production of cytokines and chemokines in THP-1 monocytic cells and PBMs at mRNA and protein level, which is against the current understanding of the pathogenesis of neonatal meningitis. However, most of the studies that determine the production of proinflammatory cytokines in experimental models were performed after 24 h of bacterial challenge [26 ]. Therefore, the observed suppression of proinflammatory cytokines by E. coli K1 may reflect a situation at the early stages of infection to temporarily delay or dampen the immune response to allow initial rounds of E. coli K1 multiplication inside monocytes. Another advantage of the altered expression of cytokines is that E. coli K1 may also avoid the neutrophil infiltration to the sites of infection. These results are distinct from the results obtained for GBS interaction with monocytes, in which GBS causes increased production of TNF-{alpha} [24 ]. Similar to our results, Toxoplasma gondii also suppresses the production of TNF-{alpha} and IL-12 in peritoneal macrophages and macrophage cell lines [27 ].

The IKK complex appears to be the major phosphorylating kinase for I{kappa}B [11 ]; nevertheless, there is some evidence that the MAPKs might be involved upstream of IKK [28 ]. It is interesting that OmpA– E. coli induced the activation of ERK 1/2, p38 MAPK, and JNK in infected monocytes, and inhibition of MAPK kinase (MEK)/ERK and p38 MAPK but not JNK resulted in inhibition of I{kappa}B phosphorylation and NF-{kappa}B activation. This suggests a role for these kinases in the activation of NF-{kappa}B by OmpA– E. coli and implies that a lack of NF-{kappa}B activation would correlate with a lack of activation of the ERK 1/2 and p38 MAPK pathways. However, we observed that infection of monocytes with OmpA+ E. coli, which does not result in significant I{kappa}B phosphorylation or NF-{kappa}B activation, still induced activation of ERK 1/2, p38 MAPK, and JNK, although the time course and extent of their activation were altered compared with that seen in OmpA– E. coli-infected cells. Thus, although ERK 1/2 and p38 MAPK appear to play a role in NF-{kappa}B activation in OmpA– E. coli-infected cells, it is unclear whether the differences in MAPK activation could account for the lack of NF-{kappa}B activation in OmpA+ E. coli-infected cells. It is possible that in the presence of OmpA, bacterial invasion induces additional events that counteract MAPK activation and lead to inhibition of NF-{kappa}B. Such possibility is also suggested by the fact that OmpA+ E. coli, but not OmpA– E. coli, actively suppresses the ability of monocytes to respond to stimulation by added LPS, preventing MAPK and NF-{kappa}B activation and cytokine production. It is possible that OmpA interacts with a cognate receptor on monocytes to induce a signaling event, which blocks MAPK and NF-{kappa}B activation. Alternatively, OmpA+ E. coli may induce the synthesis of a bacterial protein that can modulate signaling pathways, as suggested by the requirement for live bacteria and by the reduced ability of OmpA+ E. coli to suppress the production of cytokines and chemokines after incubation with the bacteriostatic agent chloramphenicol (unpublished results). Several pathogens inject virulence factors into target cells to inhibit kinase activation [8 ]. For example, YopJ of Yersinia pseudotuberculosis targets MEK, which is upstream of I{kappa}B phosphorylation [29 ]. T. gondii also suppresses the LPS-induced activation of NF-{kappa}B and cytokine production similar to that of E. coli K1; however, the mechanism for this appears to be preventing NF-{kappa}B nuclear localization, despite triggering phosphorylation-dependent degradation of I{kappa}B [27 ]. Additional studies are required to understand the molecular mechanisms involved in OmpA+ E. coli-induced inhibition of LPS signaling.

Several lines of evidence indicate that increased activity of NF-{kappa}B provides protection against apoptotic killing induced by different stimuli [30 31 32 ]. Suppression of NF-{kappa}B activity triggers apoptosis by bacterial pathogens such as uropathogenic E. coli, Shigella flexneri, and Yersinia enterocolitica [33 34 35 ]. Our studies have shown recently that OmpA+ E. coli induces the expression of antiapoptotic protein BclXL for the survival of monocytes and macrophages [7 ]. Thus, our current data, along with our previous studies, indicate that OmpA+ E. coli interaction with monocytes induces the down-regulation of NF-{kappa}B activity to suppress the immune response but at the same time, induces BclXL for the survival of macrophages to nullify the apoptotic signaling. Therefore, a fine balance between proapoptotic and antiapoptotic signals is crucial for survival of E. coli inside phagocytic cells.

In summary, our study demonstrates that OmpA+ E. coli suppresses the production of cytokines and chemokines from monocytes by inhibiting the NF-{kappa}B and MAPK activation, which may enable the bacteria to find a temporary, safe haven within monocytes, in a way, allowing parasite persistence for longer periods for multiplication.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant AI40567 and by Childrens Hospital Los Angeles Career Development Research Fellowship (to S. K. S). We are grateful to Martine Torres for critical reading of the manuscript and helpful suggestions. We also thank Sheng-he Huang for providing IbeA–, IbeB–, and Sfa– E. coli strains.

Received September 15, 2004; revised February 25, 2005; accepted April 13, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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