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Department of Pathology, Childrens Hospital Los Angeles1 and University of Southern California Keck School of Medicine, Los Angeles2 , CA 90027, USA
Correspondence
Stacy M. Townsend
stacy.townsend{at}ntu.ac.uk
Received 27 February 2006
Accepted 8 August 2006
Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; FACS, fluorescence activated cell sort; i.c., intracranial; IL, interleukin; i.p., intraperitoneal; PMA, phorbol 12-myristate 13-acetate.
The GenBank/EMBL/DDBJ accession number for the fliP sequence of Citrobacter koseri mutant SMT350 determined in this study is DQ233671.
Present address: School of Biomedical and Natural Sciences, The Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK. 
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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For this study, our objective was to identify and characterize C. koseri mutants demonstrating attenuated recovery from macrophages, indicating a reduction in uptake or persistence. Several such mutants were identified, including a fliP mutant, SMT350, which was non-motile with reduced uptake by macrophages but hypervirulent in vivo. In addition, we showed that flagellin influences the IL-10/IL-12 cytokine response during infection and may be an important factor contributing to the chronic nature of this disease.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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U937 macrophage cultivation and gentamicin protection assay. U937 macrophages were obtained from the ATCC (CRL-1593.2) and seeded into 75 ml tissue culture flasks (Sundström & Nilsson, 1976). Cells were cultivated in RPMI 1640 with 2 mM L-glutamine, modified to contain 10 mM HEPES, 1 mM sodium pyruvate, 2.5 mM glucose and 1.8 mM sodium bicarbonate, and supplemented with 10 % fetal bovine serum (Sundström & Nilsson, 1976). At least 24 h prior to infection, cells were treated with 0.1 µg phorbol 12-myristate 13-acetate (PMA; Sigma) ml–1, placed in tissue culture plates and left at 37 °C, 5 % CO2 to adhere and become activated. Cells were gently washed with RPMI 1640 to remove residual PMA and fresh medium was added prior to infection. U937 human macrophages were infected at an m.o.i of 10 for 45 min at 37 °C in 5 % CO2. After a 45 min incubation period, macrophages were resuspended in U937 medium supplemented with 100 µg gentamicin ml–1 and incubated for an additional 45 min at 37 °C in 5 % CO2. Macrophages were then washed twice, lysed with 0.5 % Triton X-100, serially diluted and plated to determine the number of intracellular bacteria at various time points (Zaidi et al., 1996). For extended assays (persistence assays), cells were replenished with fresh medium containing 10 µg gentamicin ml–1 (above the MIC). Trypan blue exclusion staining indicated that macrophage viability ranged from 80 to 95 % and was maintained for at least 72 h. For persistence assays, results for each time point are presented as the log of the percentage of inoculum that was intracellular. All assays were performed in triplicate at least two times.
Construction of C. koseri gfp fusion mutant bank.
To construct banks of random gene fusion mutants within C. koseri, three independent tri-parental matings containing C. koseri strain SMT319 (recipient strain), SM10
-pir pLOFKmgfp (transposon donor strain) (Stretton et al., 1998) and DH5
with helper plasmid pRK2013 were performed, which gave rise to exconjugates (Afendra & Drainas, 1987). Briefly, approximately 108 cells of each bacterial strain were added to 5 ml 10 mM MgSO4 and filtered through a Millipore 25 mm diameter type HAWP 0.45 µm sterile membrane. After filtering, the membrane was placed (cell side up) on the surface of a Luria–Bertani (LB) agar plate and incubated for 5 h, transferred to an LB agar plate containing 100 µm IPTG (to increase expression of the transposase) and incubated overnight (Stretton et al., 1998). The filter was then resuspended in 5 ml 10 mM MgSO4 and 200 µl was plated on to LB agar supplemented with 50 µg kanamycin ml–1 and 15 µg nalidixic acid ml–1 to select for exconjugates.
Differential fluorescence induction screening and characterization of C. koseri gfp fusion mutants. Exconjugates were isolated from selective agar plates and grown to exponential phase in experimental media. Bacteria were subjected to an initial fluorescence activated cell sort (FACS) from which the bottom 10 % of non-fluorescent bacteria was collected, thus removing constitutively fluorescent mutants. As described above, bacteria were added to monolayers of PMA-stimulated U937 human macrophages and incubated for 1 h. The macrophages were washed twice and incubated with experimental medium supplemented with 100 µg gentamicin ml–1 for 1 h. The medium was then replaced with medium supplemented with 10 µg gentamicin ml–1 and incubated for an additional 3 h. Macrophages were washed extensively with RPMI 1640 and lysed with 0.5 % Triton X-100. The resultant bacterial suspension was subjected to a second FACS from which the top 10 % of fluorescent cells were selected for further characterization.
To determine the mean fluorescence induction (MFI) of individual mutants, bacteria were incubated with macrophages as described above and subjected to FACS analysis. MFI was calculated as the ratio of fluorescence of U937-associated bacteria compared with bacteria grown in experimental medium alone.
FACS analysis was performed using the FACSDiVa and the FACSDiVa software program (Becton Dickinson). FACS parameters were set using SMT320 as a positive control. Sort gates were set for each mutant bank by applying region markers on the histogram of the logarithmic green fluorescence channel and sorting the top 10 % and bottom 10 % of the fluorescent populations.
Qualitative screening to identify mutants with attenuated uptake and persistence.
Qualitative assessment of uptake and persistence was performed as follows. Mutant (C. koseri : : Tn10gfp) and control (E. coli K1 E44 and K-12, and C. koseri SMT319) strains to be analysed were grown in 100 µl U937 medium in 96-well plates in duplicate and incubated at 37 °C overnight. PMA-stimulated macrophages (
3.0x105 cells per well) were prepared in 96-well plates using only rows 1 and 5 of the 96-well plates. A multichannel pipette was used to inoculate 1 µl bacterial cultures on to macrophages. Uptake of bacteria was allowed to occur for 45 min at 37 °C with 5 % CO2. The medium was replaced with 200 µl U937 medium supplemented with 100 µg gentamicin ml–1 and incubated for 1 h. The medium was then replaced with 200 µl U937 medium supplemented with 10 µg gentamicin ml–1 and incubated overnight. Macrophages were washed twice with Hanks' buffered salt solution, lysed with 40 µl 0.5 % Triton X-100 for 5 min and 20 µl dH2O was added. Serial dilutions were performed by transferring 15 µl bacterial solution to the lower wells containing saline (i.e. 135 µl to rows 2, 3 and 4) and 200 µl of soft agar was then placed into each well (avoiding the production of bubbles). The plate was incubated overnight at room temperature. Visual inspection of bacterial growth readily determined potential 10-fold differences among strains.
Animal infections. Timed pregnant (E14) Sprague–Dawley rats (Charles River Laboratories, Raleigh, NC, USA) were obtained and gave birth in our vivarium after a 21-day gestation period. Litters averaged 12 pups and were kept with the mother in an opaque, polypropylene cage under a small animal isolator. Two- to 3-day-old rat pups were anaesthetized with isoflurane and inoculated with either 107 c.f.u. (106 c.f.u. SMT350 for 24 h serum studies) in PBS via an intraperitoneal (i.p., 0.1 ml) or 103 c.f.u. in PBS via an intracranial (i.c., 0.002 ml) route. Purified flagellin was obtained by acid denaturation and 300 ng was injected i.p. for rat serum studies. Briefly, the pellet from a small volume of an overnight culture of wild-type C. koseri was resuspended in 150 mM NaCl and 10 mM HCl and incubated at room temperature for 30 min to depolymerize the flagellin. The resulting supernatant was further clarified by centrifugation at 100 000 g for 90 min and concentrated via acetone precipitation. A bicinchoninic acid protein assay kit was used to quantify the flagellin concentration. i.c. inoculations were administered through a burr hole produced using a 26-gauge needle at coordinates approximately 5 mm caudal to the right eye and 2 mm right of the sagittal suture. A 33-gauge single internal cannula attached to a Hamilton 1801RN 10 µl syringe, with a 22-gauge needle via 24-gauge standard wall tubing, was used to administer the dose. The needle was inserted perpendicularly into the right parietal area, at a depth of approximately 2 mm from the external surface, and 2 µl was injected over 1 min. The needle was then carefully retracted. Rat pups were anaesthetized at the indicated time points and blood and CSF samples were collected aseptically via intracardiac and cisterna magnum punctures, respectively, as described previously (Kim et al., 1992). CSF and blood samples (10 µl) were inoculated in LB and plated on to agar plates with appropriate antibiotic selection. Rats were then euthanized and whole brains were removed. All animal experiments were performed according to protocols approved by the Childrens Hospital Los Angeles Institutional Animal Care and Use Committee.
Histopathology. Whole brains of infected neonatal rats were fixed in 10 % buffered formalin, routinely processed and paraffin embedded. Coronal sections of 4–5 µm were cut and stained with haematoxylin and eosin (H&E). Histological analysis was completed by a pathologist (I. G.-G.) who was blinded to the inoculums and time post-infection of each rat at the time of sacrifice. Animals infected with PBS were analysed as controls.
Sequence analysis. A Southern blot of SMT350 EcoRI-digested chromosomal DNA was hybridized with a non-radioactive gfp probe generated using the Prime-It Fluor Fluorescence Labelling kit (Stratagene). A 5 kb fragment was identified and cloned into pUC19. Sequencing was performed using the inverse PCR primers KNout2-F (5'-GGTATTGATAATCCTGATATG-3') and gfp1.5-IC (5'-CTTCTCCTTTACTCATATGTATATC-3') and primer walking. Searches for sequence homology were performed using BLASTX.
Quantification of rat IL-10 and IL-12 serum cytokine levels following i.p. inoculation. Commercial ELISA kits (Biosource) were used to measure rat pup IL-10 (sensitivity <5 pg ml–1) and IL-12 p40 (sensitivity <3 pg ml–1) concentrations in rat serum isolated 12 and 24 h after i.p. injection of C. koseri wild-type or mutant strain. E. coli 0111 : B4 LPS (Sigma) was used as a positive control. The manufacturer's instructions for each ELISA were followed. However, the amount of serum was diluted 1 : 10 and this was taken into account during the final calculations. Each sample was run in duplicate wells and the optical density values were averaged. Cytokine standards supplied by the manufacturers were used to generate standard curves.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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2000 C. koseri transposon insertion mutants were isolated that showed induction of gfp expression when the bacteria were within macrophages. These mutants were then screened for their ability to persist within macrophages. Upon qualitative analysis, seven of these mutants were found to be attenuated for uptake and/or persistence within macrophages. However, mutants that absolutely could not be taken up or survive within macrophages were not identified in this study.
Quantitative gentamicin protection assays were performed on the seven mutants to characterize their ability to be taken up by and persist within human macrophages. Results from these assays revealed that there were various in vitro mutant phenotypes (Fig. 1
). Mutant strains SMT324, SMT325 and SMT356 maintained wild-type uptake levels but were attenuated for intracellular replication (Table 1). Mutant strain SMT332 was attenuated for uptake and intracellular replication within macrophages; SMT336 and SMT350 were attenuated for uptake alone (Table 1). Mutant strain SMT335 showed reduced uptake and was unable to persist within macrophages (Table 1). All mutants were susceptible to gentamicin. Southern blot analysis (using the gfp gene as a probe) ruled out siblings and confirmed that single transposon insertions were responsible for the observed phenotypes (data not shown).
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). SMT332 showed the highest increase in MFI. The MFI was low for SMT335 (1.6) and moderate for SMT356 (2.3); however, both had fusions interrupting genes necessary for intracellular replication as these strains did not replicate within macrophages (Table 1). In contrast, SMT350 consistently had the lowest MFI with an intracellular generation time of 8.9±1.5 h (Table 1). SMT350 was also non-motile. Previous flagellar mutants of Salmonella enterica serovar Typhimurium have exhibited faster net growth intracellularly, although invasiveness was attenuated; however, our rate was not significantly different to that of wild-type (Schmitt et al., 2001). SMT324, SMT325 and SMT332 had intracellular generation times significantly higher than the wild-type (Table 1). Furthermore, mutant strains SMT332, SMT335, SMT336 and SMT350 were taken up between 6- and 526-fold less than the wild-type (Table 1). All strains except SMT335 were able to persist for up to 72 h within macrophages. Persistence was maintained when % survival±SEM was within 100 % at 48 h. SMT335 failed to persist and suffered an 83±6 % reduction in intracellular c.f.u. after 24 h, which was further reduced to 96±1 % at 48 h, comparable to that observed for the negative control E. coli K-12, which gave values of 80 and 98 % reduction, respectively (data not shown).
In vivo characterization of mutants deficient in macrophage uptake and persistence
C. koseri is specifically noted for its unique ability to cause brain abscesses in neonates (Kline & Kaplan, 1987; Townsend et al., 2003). To determine the consequence of deficient macrophage uptake and persistence on brain abscess formation, the ability of each strain to induce brain infection and/or abscess formation following i.c. inoculation was tested. At least six 2–3-day-old rat pups were injected i.c. with 103 c.f.u. of each strain and independently analysed histologically for brain abscess formation. A Kaplan–Meier survival curve was used to analyse survival time (SPSS). The mean survival time of 2-day-old rats injected i.c. with 103 c.f.u. of wild-type was 7.8±1.0 days (Fig. 2). Interestingly, SMT350 had a mean survival time of 2.33±0.42 days, whereas the survival time for SMT356 was indefinite because all rats survived the i.c. challenge (n=11). All other mutant survival times were similar to that of the wild-type (Table 1).
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depicts H&E-stained sections from neonatal rats infected by i.c. inoculation of wild-type C. koseri (Fig. 3a–c), SMT350 (Fig. 3d–i), SMT356 (Fig. 3j) and SMT336 (Fig. 3k). Severe bilateral ventriculitis and ventriculomegaly ensued 5 days after i.c. infection with wild-type C. koseri. Extension of the inflammation into the white matter directly preceded brain abscess formation (Fig. 3a). Brain abscess formation coincided with the accumulation of inflammatory cells such as macrophages and neutrophils and the destruction of the ventricular wall (Fig. 3b). In addition, C. koseri-filled macrophage and inflammatory cells within the white matter adjacent to the ventricle wall may have disseminated the infection to the surrounding tissue (Fig. 3c). In contrast, ventriculitis and ventriculomegaly ensued 3 days after i.c. infection with SMT350. Inflammation within the ventricle and white matter included macrophages, neutrophils, lymphocytes, plasma cells, Russell bodies and free bacteria (Fig. 3d–i). However, the inflammation seemed less intense than that following wild-type infection and consisted of different cell types, predominantly lymphocytes and neutrophils, as opposed to macrophages, which usually respond later during infection (Fig. 3d–i). The Russell bodies indicated a massive reaction to an antigen such that the antibodies produced accumulate in the cytoplasm of the plasma cells forming Russell bodies (Fig. 3e–h). SMT356 infections did not develop brain abscesses or disease, with the only traces of insult indicated by pigmented macrophages located in the ventricle, suggesting that haemorrhage had occurred within the previous 4–5 days (Fig. 3j). In addition, free and intracellular bacteria were observed markedly less frequently than with either wild-type or the other mutants at the corresponding day post-infection. The c.f.u. recovered from i.c.-infected pups (3 days old) at 7 days post-infection averaged 4.9x1010 c.f.u. (g brain tissue)–1 (n=5), whilst only 45 c.f.u. (g brain tissue)–1 (n=6) was recovered from SMT356-infected pups (data not shown). SMT356 exhibited a drastically reduced mortality rate associated with reduced recovery of c.f.u. in the brain tissue and the absence of brain abscess formation. SMT335 was attenuated in intracellular replication within human macrophages, although it maintained full virulence in vivo (Table 1) and maintained wild-type brain pathology (data not shown). Other mutants (such as SMT336) also had a brain pathology similar to that of the wild-type following i.c. injection, regardless of in vitro phenotype (Fig. 3k), suggesting that the presence of the transposon itself did not directly affect brain pathology. Consistently, the most comparable aspect of the histology, causing a considerable amount of tissue damage in addition to periventricular brain abscesses and ventriculitis, was the development of oedema and consistent formation of perivascular fluid cysts within the white matter not adjacent to abscess material.
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). Weaker amino acid identity was observed to enteropathogenic E. coli EscR (39 %) and Salmonella Typhimurium SpaP (34 %) and SsaR (38 %), which are type III secretion proteins important for the translocation of virulence factors (Galán & Collmer, 1999). Further sequence analysis revealed that fliP was one of five contiguous genes cloned having homology to E. coli fliNOPQR (Malakooti et al., 1994). The genetic organization and open reading frame orientation of these genes were identical to the E. coli fli flagella operon, which is important in the complete biosynthesis of functional flagella (Fig. 4). The fliP gene encodes an integral membrane protein associated with the basal body of the flagella structure. As the fli operon is coordinately regulated by a promoter upstream of fliL, expression of fliR and fliQ is lost via polar effects, as the transposon insertion occurs upstream in fliP (Malakooti et al., 1989). As SMT350 was the result of fliP disruption, a gene encoding an essential component of a type III secretion system for flagellin secretion, we examined the motility phenotypes of each mutant. Only SMT350 lacked motility, as assessed by a 0.3 % soft agar motility assay (Table 1 and Fig. 5a).
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). Using this approach, two revertant motile strains (SMTR1 and SMTR2) were isolated. Furthermore, gentamicin protection assays confirmed that the ability of these revertant strains to be taken up and replicate within macrophages had been restored to wild-type levels (data not shown). Interestingly, SMT350 caused mortality in infant rats before brain abscesses could occur (within 3 days), with a marked reduction in the presence of infiltrating macrophages, and a notable increase in extracellular bacteria compared with wild-type was observed in this study. Similarly, others have documented reduced polymorphonuclear influx in response to non-flagellated mutants of Salmonella (Schmitt et al., 2001). Although the migration of macrophages to the site of inflammation is understood to be non-antigen specific, these observations indicate that the expression of flagella or motility contributes to the establishment of chronic infection. We observed that non-motile (non-flagellated) SMT350 exhibited significantly reduced uptake in vitro and in vivo, resulting in accumulation of extracellular bacteria. Similarly, Salmonella Enteritidis lacking flagella is significantly attenuated in its ability to invade murine, avian and human cell lines (Lockman & Curtiss, 1990; Van Asten et al., 2000). Our data lend additional support to the hypothesis that flagella play a role in the process of invasion. It is also possible that flagella may mask a more toxic factor that is exposed when the flagellum structure is not expressed. Reduced uptake by macrophages would amplify this by increasing the exposure of the antigen, with the majority of the bacterial population remaining extracellular. This was supported by the observation that Russell bodies (accumulation of immunoglobulin proteins due to overexpression within plasma cells) were abundant following SMT350 infection and absent during wild-type infection. Moreover, the histology revealed a pattern of inflammation (Russell bodies) that was inconsistent with what is observed during LPS-induced shock.
Serum cytokine levels in rat pups following exposure to C. koseri
To determine whether mutant strains deficient in macrophage uptake and persistence differentially affected cytokine expression influencing the development of type 1 and type 2 immune responses, serum IL-10 and IL-12 levels were analysed at 12 and 24 h after i.p. infection of rat pups. At 12 h post-inoculation, only the wild-type induced IL-10 expression (281±18 pg ml–1) compared with mutants SMT350 and SMT356, where no expression was detected (Fig. 6a). IL-10 expression in response to mutant SMT350 was delayed and reduced threefold but detected at 24 h (90±81 pg ml–1) (Fig. 6a). Purified flagellin (300 ng) was also injected i.p. to ascertain the influence of flagellin on cytokine expression in vivo and IL-10 serum levels were found to increase significantly (4401±1068 pg ml–1) at 12 h post-treatment and then dropped to 302±21 pg ml–1 at 24 h post-treatment (Fig. 6a). LPS is also known to induce IL-10 and IL-12 expression and was tested as a positive control. Although IL-10 expression was dominant at 12 h post-inoculation, IL-12 expression increased twofold by 24 h and was favoured in comparison with IL-10 expression in all strains.
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The current cytokine studies showed that IL-10 expression occurred early during infection (within the first 12 h), which may initially favour a type 2 immune response to wild-type C. koseri infection. Comparatively, mutant strains SMT350 and SMT356 displayed differential IL-10 expression, which was delayed and reduced or absent, respectively. Further, C. koseri flagellin was shown to induce robust IL-10 expression in neonatal rats 12 h after i.p. injection that subsided within 24 h. In this study, it has been shown that mutant strain SMT350 exhibits delayed and reduced IL-10 expression and a hypervirulent phenotype in vivo. This indicates that flagella may possess immunosuppressive properties such that initial recognition of flagella alters the conventional mechanism for resolving C. koseri infection. These studies show that Citrobacter flagellin induces early IL-10 expression and may alter the host response to favour chronic infection, resulting in brain abscess formation. Previous studies have shown that various pathogens can deregulate the host cytokine response as a survival mechanism during infection (Lucey et al., 1996; Wilson et al., 1998). For example, when human macrophages are infected with Mycobacterium avium, expression of the immunoregulatory cytokine IL-12 (type 1 cytokine) is suppressed, impairing the immune response that is pivotal in resistance to intracellular infections (Wagner et al., 2002). Furthermore, it has been suggested that other highly adapted intracellular-replicating organisms inhibit the type 1-specific response by suppressing expression of its mediating cytokines (i.e. IL-12) and/or inducing cytokines that mediate a type 2-specific response (i.e. IL-10) (Grazia Cappiello et al., 2001; Wagner et al., 2002; Wilson et al., 1998). Supporting studies have used lysates from E. coli and Citrobacter spp. and demonstrated inhibition of IL-12 and IL-4 production by lymphoid cells (Malstrom & James, 1998). This suggests that bacterial factors may be responsible for the induction or suppression of cytokine expression. Furthermore, it has been shown by others that Fc
receptor ligation induces IL-10 upregulation, precluding IL-12 expression (Grazia Cappiello et al., 2001; Sutterwala et al., 1998). Our previous studies have documented that FcRI is the major facilitator of opsonized C. koseri uptake in macrophages (Townsend et al., 2003). This suggests a possible mechanism enabling an intracellular signal to induce a cytokine response by cell contact alone.
The ability of C. koseri to invade and persist within host cells is an important step in the development of chronic CNS infection. In this study, we have shown that the fliP mutation causes a significant change in pathogenesis and in the host immune response during C. koseri infection and reduces the uptake of C. koseri into human U937 macrophages in vitro. However, the fliP mutant strain SMT350 is hypervirulent, essentially converting a chronic disease into an acute infection. Moreover, we have established that C. koseri intracellular replication within macrophages is not sufficient for brain abscess formation to occur, as the attenuated intracellular replication phenotype was separable from brain abscess formation. Thus, mutants with attenuated abilities to replicate within macrophages were still able to cause brain abscess formation during experimental i.c. infection of neonatal rats (i.e. SMT332 and SMT335). Cytokine studies have shown that the immunosuppressive cytokine IL-10 is expressed during the first 12 h of serum exposure to purified flagellin, whilst this expression was delayed and reduced in the fliP mutant SMT350. Taken together, flagella expression may play a pivotal role in influencing the host immune response to C. koseri infection by inducing an environment conducive to maintenance of chronic infection. The specific role(s) of flagella during the pathogenesis of C. koseri CNS infection is multifaceted and requires further study.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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stimulated macrophages: regulation by endogenous tumor necrosis factor- and by IL-10. Int Immunol 6, 693–700. receptor ligation. J Immunol 166, 4498–4506. production. Proc Natl Acad Sci U S A 93, 3193–3198. receptor type I. J Exp Med 188, 217–222.). Curr Opin Immunol 9, 17–23.[Medline]This article has been cited by other articles:
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  S. Liu and T. Kielian Microglial Activation by Citrobacter koseri Is Mediated by TLR4- and MyD88-Dependent Pathways J. Immunol., November 1, 2009; 183(9): 5537 - 5547. [Abstract] [Full Text] [PDF]
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6) were inoculated i.c. with 103 c.f.u. of each strain and monitored daily for clinical signs of disease or death for 11 days. Rats inoculated with the mutant strain SMT356 or PBS (n=4) had no fatalities.
WT, n=2), wild-type C. koseri (WT, n=6), mutant C. koseri SMT350 (n=7) or SMT356 (n=7). Rats administered saline (negative control) did not produce detectable levels (not shown). Data represent the mean±SEM of duplicate samples.