Gene Expression Pattern in Human Brain Endothelial Cells in Response to Neisseria meningitidis

Bacterial strains. Neisseria meningitidis strain MC58 (B15:P1.7,16b) and the MC58 siaD unencapsulated mutant (60) were routinely cultured in proteose-peptone medium supplemented with 1% polyvitex (bioMérieux, Lyon, France) to the mid-logarithmic phase and diluted to approximately 1 × 10⁷ CFU in RPMI 1640 medium (Biochrom AG, Berlin, Germany) supplemented with 10% heat-inactivated (30 min at 56°C) human serum for all cell culture experiments.

Endothelial cell culture.HBMEC were isolated from a brain biopsy of an adult female with epilepsy, transfected with the simian virus 40 large T antigen, and cultured as described previously (55, 56). HBMEC maintained their morphological and functional characteristics of primary brain endothelial cells for up to 15 passages. Cells were cultured in RPMI 1640 medium, supplemented with fetal calf serum (10%), Nu serum IV (10%), vitamins (1%), nonessential amino acids (1%), sodium pyruvate (1 mM), l-glutamine (2 mM), heparin (5 U ml⁻¹) (all reagents were from Biochrom, Berlin, Germany), and endothelial cell growth supplement (30 μg ml⁻¹) (Habor Costar Corporation, Cambridge, MA). The cells were maintained in T25 flasks (Corning Costar Corporation, Cambridge, MA) coated with 0.2% gelatin in a humid atmosphere at 37°C with 5% CO₂. For microarray experiments and real-time PCR (LightCycler; Roche), the cells were used at passage 10. For further experiments, passages 10 to 15 were used.

HBMEC infection.For microarray hybridization and real-time PCR experiments, HBMEC were split and seeded onto T75 flasks at a density of 0.5 × 10⁷ cells per flask and cultured for 48 h. Two hours prior to infection, confluent cell monolayers were washed three times with RPMI 1640 medium. HBMEC were infected with either wild-type (WT) strain M58 or the MC58 siaD capsule mutant strain at a multiplicity of infection (MOI) of 10. Control cells received fresh RPMI 1640 supplemented with 10% heat-inactivated human serum. After 4 hours and 8 hours of infection, bacteria were removed and HBMEC were washed three times with phosphate-buffered saline (PBS; Biochrom AG, Berlin, Germany), trypsinized as described before (12), resuspended in ice-cold PBS, and centrifuged at 1,000 × g for 5 min at 4°C. The snap-frozen pellets were then stored at −70°C for RNA isolation.

For fluorescence-activated cell sorter (FACS) analysis and enzyme-linked immunosorbent assay (ELISA) experiments, HBMEC were seeded onto 12-well plates (Corning Costar Corporation, Cambridge, MA) at a density of 1 × 10⁵ per well 2 days prior to the experiments. The cells were washed and infected as described above. After 8, 16, and 24 h of infection, supernatants were collected for cytokine assessment, centrifuged at 10,000 × g for 5 min at 4°C, and frozen for storage at −70°C. For FACS analysis, cell monolayers were washed twice with RPMI 1640 medium. Cells were detached with 0.25% trypsin and washed once with PBS following specific FACS protocol.

RNA isolation, cDNA preparation, and hybridization.Total RNA was extracted with saturated phenol-chloroform and precipitated in isopropanol, and pellets were washed with 80% ethanol, dried, and resuspended in RNase-free H₂O. Subsequently, total RNA was treated with DNase in the presence of 1 U ml⁻¹ anti-RNase (Ambion) and 45 μg of total RNA was used for poly(A)⁺ RNA enrichment using a BD Atlas Pure Total RNA labeling system kit (BD Bioscience Clontech, Palo Alto, CA) and [³³P]-dADP (Amersham Pharmacia, Hong Kong). Labeled cDNA was separated from unincorporated nucleotides with NucleoSpin extraction spin columns following hybridization onto prerinsed plastic microarrays at 60°C for 14 h according to the manufacturer's instructions (BD Atlas plastic human 12K microarray kit; BD Bioscience Clontech, Palo Alto, CA). The microarrays were washed twice with 2× SSC-0.1% sodium dodecyl sulfate (SDS) (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and twice with 0.1× SSC-0.1% SDS for 5 min at 60°C in all cases and then twice with 0.1× SSC at 30°C for 5 min each time.

Array analysis.For the cDNA microarray experiments, BD Atlas plastic human 12K microarrays (BD Bioscience Clontech) were used. The array contains a selection of 11,835 human genes (oligonucleotides), including 9 housekeeping genes, cDNA synthesis controls, and negative controls (various phage λ sequences) printed in duplicate on a plastic support surface. Equal amounts of labeled probes from MC58- and MC58 siaD strain-infected and uninfected cells were hybridized to the microarrays according to the manufacturer's instructions. After the washing procedure (twice with 2× SSC-0.1% SDS and twice with 0.1× SSC-0.1% SDS for 5 min at 60°C in all cases and then twice with 0.1× SSC at 30°C for 5 min each time) was completed, the arrays were exposed with a low-energy phosphorimaging screen (Molecular Dynamics, CA) overnight and for 3 days. The signal intensities were detected with Imager Analyzer FLA-3000 using “Image Reader” soft (Fuji Photo Film Co., Ltd.) at 50-μm resolution. The intensities of cDNA double spots from infected and uninfected cells were compared, and the data were exported as Excel files for further analysis. The probes were stripped from the arrays following the manufacturer's recommendations and then rehybridized with newly labeled probes. The resulting expression profiles were virtually identical to those from the first hybridization, but with only 70% of the original intensities. This phenomenon has already been described by other users of this cDNA microarray platform (30). We also presented data only from the first experiment, in agreement with published data.

Arrays were further analyzed using AtlasImage 2.7 software (Clontech) according to the manufacturer's guidelines. Using AtlasImage 2.7 software, the arrays were aligned with a BD AtlasImage grid template (BD Clontech) automatically and then fine-tuned for each gene by using manual adjustment options. The background was calculated based on the median intensity of the “blank spaces” between different panels of the array (default method of calculation), and the raw signal intensity of each spot was measured. A raw intensity (before normalization) of twofold over background was taken as an indication that a gene was expressed at a significant level. Using this criterion, we found 1,628 genes expressed in HBMEC at 4 h and 1,489 genes expressed at 8 h after infection with encapsulated meningococci (strain MC58) (Table 1). A list of all of the expressed genes is presented in the supplemental material. For comparison of the expression patterns of MC58-infected cells and uninfected cells, the signal intensities were normalized by the global normalization sum method, which is best suited for the comparison of two similar tissue samples. Signal values for arrays hybridized with MC58-infected cDNA were normalized with respect to those for arrays reprobed with cDNA of uninfected cells. The adjusted signal intensities of each of the individual cDNA spots were compared for infected and uninfected cells. The ratios (numerical values in Tables 2, 3, and 4) were calculated as the adjusted intensity of array 2 (infected HBMEC) divided by the adjusted intensity of array 1 (uninfected cells) according to the manufacturer's guidelines. Differences are estimated when a gene signal either in array 1 or in array 2 is at background level. Instead of using numerical values, we indicated these genes as up-regulated (↑) or down-regulated (↓) in infected HBMEC. The results were saved in a tab-delimited format and opened by Microsoft Excel for further analysis. To further search for significant changes of gene classes, we used GoMiner, a program that classifies genes into biological categories and calculates the probability of over- and underrepresentation within a particular group, compared with the master list by use of Fisher's exact test (65).

Real-time PCR.Differential gene expression data were validated using an SYBR green-based real-time quantitative PCR method. HBMEC were infected with the MC58 wild-type strain and the MC58 siaD mutant strain or exposed to RPMI medium supplemented with 10% heat-inactivated human serum, and at 4 h and 8 h postinfection (h p.i.), cell monolayers were washed and total RNA was extracted using an RNeasy mini kit (QIAGEN). The total RNA content was determined spectrophotometrically, and equal amounts of RNA were treated with RNase-free DNase (Roche). Total RNA (2 μg) was reverse transcribed with random nonamers (2.5 μΜ) using Omniscript reverse transcriptase (4 U ml⁻¹) according to the manufacturer's instructions.

For quantitative PCR, SYBR green master mix (Roche) was used according to the manufacturer's instructions.

Primers specific to immediate-early response gene 1 (forward, 5′-GAA CTG CGG CAA AGT AGG AG-3′; reverse, 5′-GAC AGT CGC TCC GTG ACA GC-3′), bak (forward, 5′-CGA CAT CAA CCG ACG CTA TG-3′; reverse, 5′-GCA ATG CAG TGA TGC AGC ATG-3′), bad (forward, 5′-TGG ATG ACC TCG ATG ATG AAG-3′; reverse, 5′-GCA CAG CAA CGC AGA TGC G-3′), asp (forward, 5′-CTG ACT CAC ATA CAG TAG ATC-3′; reverse, 5′-GGA TAC TAT GAT TTG ACA GGC-3′), and req (forward, 5′-CGT GGC TTC ACA CGC ACC-3′; reverse, 5′-AAG TGG GAG TGG GCA TAG TG-3′) were designed and used for amplification of 261-, 244-, 266-, 222-, and 208-bp amplicons, respectively.

Preliminary experiments showed that the β-actin transcription level was not affected under the experimental conditions to which the cells were subjected. The results were analyzed according to the method described by Pfaffl (46). For this mathematical method, the efficiency (E) of each PCR is calculated from the slopes of cDNA input versus the crossing points (CP) plot as follows: E = 10^(−1/slope). The relative expression ratio of the gene of interest to the target gene was calculated with respect to the reference gene (β-actin gene) by comparing HBMEC infected with meningococci and untreated control cells. The ratio was calculated as follows: (E_target)^{ΔCP target (control−sample)}/(E_reference)^{ΔCP reference (control−sample)}.

Flow cytometry.Antibodies to intercellular adhesion molecule 1 (ICAM-1) (fluorescein isothiocyanate conjugated; Biosource, Camarillo, CA) were purchased and used in flow cytometric analysis to examine the protein expression in HBMEC. Briefly, following infection cells were blocked with 3% bovine serum albumin for 30 min, and cells were stained with anti-ICAM-1 (1:20) for 45 min at 4°C. Following staining, cells were fixed with 1% paraformaldehyde for 15 min and analyzed by fluorescence measurement. Mouse immunoglobulin G1 fluorescein isothiocyanate conjugate (Biosource, Camarillo, CA) was used as described above and served as an isotype control. For apoptosis detection, an annexin V-biotin kit (Immunotech, France) was used. Following infection, cells were collected, washed, and stained with 5% annexin V-biotin solution in 1× binding buffer (PBS containing 5.5 mM d-glucose and 0.5% bovine serum albumin) for 10 min. Subsequently, cells were fixed with 1% paraformaldehyde (methanol free; Polysciences, Warrington, PA) for bacterial killing. Cells were washed once with annexin V-biotin solution in 1× binding buffer and incubated with 20 mg ml⁻¹ streptavidin-phycoerythrin conjugate for 20 min. All staining steps were conducted on ice. Flow cytometric analyses were performed on a FACSCalibur flow cytometer (Becton Dickinson) and analyzed using CellQuest software, version 3.1.

Cell lysate preparation and Western blotting.At various time points of infection, cells were washed three times with PBS. Washed cells were lysed in 1× SDS sample buffer directly on a plate, collected, and boiled for 5 min. Cellular proteins were resolved by 10% (Akt and AMP-activated protein kinase α [AMPK-α]) or 15% (caspase 3) SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After being blocked for 1 h in Tris-buffered saline-Tween (25 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20) containing 6% dry milk (Bio-Rad, Munich, Germany), membranes were incubated with primary antibodies at 4°C overnight. The following antibodies were used: rabbit anticleaved caspase 3 (Asp175), anti-phospho-AMPK-α (Thr1729), and anti-phospho-Akt (Ser437) (Cell Signaling Technology Inc., Beverly, MA). Membranes were washed briefly and incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Bio-Rad, Munich, Germany) secondary antibodies (1:3,000 in 3% dry milk-Tris-buffered saline-Tween 20) for 50 min at room temperature. Immunoreactivity was detected using an ECL or ECL Plus detection kit (Amersham Pharmacia Biotech, Freiburg, Germany). To detect endogenous levels of activated proteins, blots were stripped and reprobed with antibodies specific to the nonphosphorylated form of the protein of interest. The protein bands on X-ray films were scanned by using a StudioStar imaging densitometer, Agfa, and the intensities of bands were analyzed using NIH Image software.

Cytokine assessment by ELISA of HBMEC supernatants.Concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the cell culture supernatants were measured by commercially available ELISA kits (AMS Biotechnology GmbH, Abington, United Kingdom). The ELISAs were performed according to the manufacturer's instructions to a sensitivity of 4 pg ml⁻¹. For all ELISA systems, the 3,3′,5,5′-tetramethyl(benzidine) (TMB) substrate reagent set (BD Pharmingen, Heidelberg, Germany) was used to detect the streptavidin-horseradish peroxidase (BD Pharmingen) reaction. All samples were assayed in duplicate. The experiments were carried out at least four times.

Statistical analysis.The statistical analysis of the results was done using Student's t test. A P value of <0.05 was considered significant, and data represent means ± standard errors of the means (SEM).

Expression profile of HBMEC induced by N. meningitidis.To investigate the effect of meningococcal infection on the transcriptome of HBMEC, we used BD Atlas plastic human 12K microarrays (BD Clontech). This cDNA-based microarray platform contains 11,835 different human genes. HBMEC were used already as a valuable tool for studying the translocation of the B-CSF barrier by meningitis-causing pathogens (1, 21, 45, 48, 60). To discriminate changes of the transcriptome, both infected and control cells were analyzed 4 h and 8 h after infection with meningococci. At 4 h p.i., bacteria reached saturation of adhesion to HBMEC with only low invasion, whereas at 8 h p.i., a maximum of bacteria was internalized (60).

These analyses revealed that 366 genes showed similar levels of expression in MC58-infected and uninfected HBMEC at 4 h p.i. (Table 1). A total of 374 genes (30% of genes with a ratio of at least twofold) were up-regulated in MC58-infected cells, and 888 genes (70%) were down-regulated. After a period of 8 h of infection, the activity of gene expression turned around: while 741 genes (74.6%) were overexpressed in MC58-infected cells, 252 genes (25.4%) were down-regulated (Table 1). A total of 496 genes showed similar levels of expression in infected and uninfected HBMEC.

The expressed genes comprise all functional categories (Table 1): transcription (100 genes at 4 h p.i. and 104 genes at 8 h p.i.; P < 0.01 as analyzed with GoMiner), membrane channel proteins and transporters (48 at 4 h p.i. and 45 at 8 h p.i.; P < 0.01), metabolism (182 at 4 h p.i. and 169 at 8 h p.i.; P < 0.01), posttranslational modification/protein folding (53 at 4 h p.i. and 47 at 8 h p.i.; P = 0.57), protein translation (99 at 4 h p.i. and 102 at 8 h p.i.; P = 0.05), intracellular transducers/effectors/modulators (145 at 4 h p.i. and 127 at 8 h p.i.; P = 0.42), and cytoskeleton/motility proteins (52 at 4 h p.i. and 52 at 8 h p.i.; P = 0.0024) were detected. A total of 552 genes (4 h p.i.) and 438 genes (8 h p.i.) that have not been functionally classified or are unclassified were also expressed at elevated levels. Further analysis revealed that at 4 h p.i., most of the genes were of low abundance (70%) and about 30% of them were expressed at levels at least 10-fold over background. At the late stage of infection, only 42% of the genes were of low abundance and 58% of them were at levels at least 10-fold over background.

Table 2 categorizes the most notable genes and their 4-h and 8-h induction values. The highest levels of induction were observed for genes encoding ribosomal proteins with levels at least 10-fold over background, such as ribosomal proteins L10a, L27, and S15a (Table 2).

Cell surface antigens.Five cell adhesion molecules were expressed at relative high levels in MC58-infected cells. Among them, the hyaluronate receptors CD44, CD98, and CD99 were the most prominent. CD44 mediates human cell-cell- and cell-extracellular matrix-binding interactions and has also been described to function as a receptor for group A streptococcus colonization of the pharynx in vivo (10). Internalization of piliated capsulated meningococci has been shown to be triggered by the formation of microvillus-like membrane protrusion, containing eszin, moesin, and the integral membrane proteins CD44 and ICAM-1 (24). Atlas plastic human 12K microarrays also contained cDNAs for ICAM-1 (CD54), and mRNA for ICAM-1 was also shown to be highly expressed with 5-fold and 18-fold expression levels over background at 4 h p.i. and 8 h p.i., respectively (Table 2), while expression in uninfected HBMEC was undetectable. To verify whether the changes in ICAM-1 mRNA levels were reflected by protein expression, flow cytometry of immunostained MC58-infected and uninfected HBMEC was performed. After 8 h of incubation, a marked increase of ICAM-1-positive events (34%) was detected (Fig. 1A and B), and 16 h after infection, 62% of events from cultures were ICAM-1 positive compared to those from uninfected cells.

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FIG. 1.

Expression of ICAM-1 on HBMEC at 8 h and 16 h after incubation with the MC58 (striped bars) and MC58 siaD (black bars) strains at an MOI of 10 in comparison to that on uninfected HBMEC (open bars). Results are expressed as means and standard errors of the means for four independent experiments. *, P < 0.05 in comparison with uninfected HBMEC; **, P < 0.01 in comparison with uninfected HBMEC; ', P < 0.05 for ICAM-1 expression on HBMEC after incubation with the MC58 siaD strain in comparison to that on cells after incubation with the encapsulated MC58 strain. For comparison, the mean fluorescence intensities for panel A are shown in panel B. FL1-H, fluorescence intensity on channel that detects emissions from fluorescein isothiocyanate.

Interestingly, a substantial level of CD98 was expressed in MC58-infected HBMEC at 4 h p.i. and 8 h p.i. CD98 acts as an important regulator of integrin-mediated adhesion (50). It is constitutively associated with β₁ integrins regardless of the activation status. We previously described that invasion of N. meningitidis in HBMEC is mediated by α₅β₁ integrins (60). In accordance with data from Plant and colleagues (47), the gene encoding the integrin β₁ chain was not up-regulated. However, levels of the integrin α₅ transcript were significantly increased (Table 2).

Intracellular transducers/effectors/modulators.Infected cells expressed genes involved in downstream signaling of α₅β₁ integrins and of other receptors such as ICAM-1 and CD44, including integrin-linked kinase (ILK), Rho GTPase-activating protein 1, and regulatory enzymes such as guanine nucleotide exchange factors (e.g., Rho Gef12) (Table 2). Two further members of the RAS superfamily (rhoG and rhoC) were highly up-regulated at 4 h p.i. and 8 h p.i. in MC58-infected cells. We furthermore observed regulated genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway: MAP kinase kinase 1 and MAP kinase kinase kinase 10 were down-regulated after infection. While MAP kinase kinase 1 regulates the extracellular signal-regulated kinase signaling pathway, MAP kinase kinase kinase 10 has been shown to function preferentially on the Jun N-terminal protein kinase signaling pathway (52). At 8 h of infection, dual-specificity phosphatases (DUSPs) 1, 5, and 14 and G protein pathway suppressor 2 (GPS2) were found to be up-regulated in infected cells, indicating expression of genes involved in negative regulation of downstream MAPK signaling pathways.

Cell signaling/extracellular communication proteins.Another finding of the comparative gene expression analysis was that low, but clearly above background, levels of multiple cytokines and chemokines were observed in MC58-infected cells (Table 2). The genes of the cytokines IL-6, TNF-α, chemokine-like factor 1, CC chemokine CCL19 (macrophage inflammatory protein 3β and EBI1 ligand chemokine), and insulin-like growth factor 1 (somatomedin C) were up-regulated in infected cells. IL-1α, insulin-like growth factor 2 (somatomedin A), inducible VGF nerve growth factor, and CC chemokine member 22 (SCYA22) were down-regulated at the analyzed time points.

IL-6 and TNF-α have already been shown to be induced by N. meningitidis upon infection of meningothelial and endothelial cells (51, 53, 64). To analyze whether changes in mRNA levels were reflected in protein expression, ELISAs were performed for the detection of IL-6 and TNF-α protein secretion. Supernatants from infected and uninfected HBMEC cultured for 16 h were analyzed, and the concentrations of both cytokines, IL-6 and TNF-α, were markedly elevated in the supernatants obtained at 8 h of infection (P < 0.05) and peaked at 16 h p.i. (Fig. 2). At 16 h p.i., the concentration of IL-6 was 5.3 times higher in response to MC58-infected cells than in response to control cells. TNF-α was not detectable in uninfected cells, but its concentration increased in supernatants from infected cells up to 96.3 ± 9.3 pg ml⁻¹ at 16 h p.i. However, genes for many other proinflammatory markers produced by epithelial or endothelial cells in response to bacterial pathogens (e.g., IL-8 and growth-related oncogenes α and β, which belong to the CXC subfamily of chemokines) were not induced in HBMEC by N. meningitidis at 4 h p.i. and 8 h p.i.

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FIG. 2.

Concentrations of IL-6 (A) and TNF-α (B) in HBMEC culture supernatant infected with either the MC58 (striped bars) or MC58 siaD (black bars) strain for 8 and 16 h. Controls (uninfected cells) are shown as open bars. Cytokine concentrations were assessed by ELISA, and the limits of sensitivity were 4 pg ml⁻¹. Results are expressed as the means ± SEM for four independent experiments carried out in duplicate; **, P < 0.01 relative to the respective control; ', P < 0.01 compared to the MC58 siaD strain.

Cell adhesion receptors/proteins.The array data represent cDNAs for a number of cell adhesion proteins that have been reported to play an important role in maintaining cellular integrity. The mRNAs that showed a decrease in MC58-infected cells were claudin 12, claudin 14, protocadherin 8, and protocadherin 17. Claudins are transmembrane proteins of tight junctions and take part in the regulation of brain endothelial permeability (35). Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules and are assumed to be anchored to the cytoskeleton (18).

Cytoskeleton/motility proteins.Our analysis also showed a strong enrichment of factors that interact with the host cell cytoskeleton (P = 0.0024). Several genes involved in the reorganization of the cytoskeleton or genes encoding motility proteins, e.g., the actin-related protein 3 (ARP3) homolog, the actin-related protein 2/3 complex, subunit 2 (p34-Arc), the mRNA of actinin alpha 1 (ACTN1), and cytoskeletal gamma actin as well as alpha 2 actin, were up-regulated in MC58-infected cells mainly at the late the stage of infection, indicating cytoskeleton rearrangements in the context of bacterial internalization. Although a number of genes involved in cell cytoskeleton and motility were up-regulated as mentioned above, two components of the host cell cytoskeleton machinery were found to be down-regulated: the vasodilatator-stimulated protein (VASP), which is involved in the regulation of actin filament assembly and cell motility, and the Wiskott-Aldrich syndrome protein (WASP).

Capsule-dependent changes in gene expression.Next, we investigated the contribution of the capsular polysaccharide to the observed changes in HBMEC gene expression. For these studies, we took advantage of an unencapsulated mutant of MC58. In this strain, the polysialyltransferase gene siaD was inactivated by insertional inactivation as described earlier (60). We adopted a reductionist approach to identify those genes whose expression was influenced by the capsule. First, we used AtlasImage 2.7 software to identify gene expression patterns that were significantly different for MC58 siaD mutant-infected cells compared to those for uninfected cells. This comparison revealed 231 genes at 4 h p.i. and 253 genes at 8 h p.i. that were expressed independent of capsule expression (Fig. 3). Analysis with GoMiner revealed an enrichment of genes involved in nucleotide biosynthesis/transcription (P = 0.02), metabolism (P = 0.01), cell motility (P = 0.02), particularly the Arp2/3 protein complex, and regulation of endocytosis (P = 0.02). In contrast, expression of 624 genes at 4 h p.i. and 449 genes at 8 h p.i. altered by the encapsulated wild-type strain MC58 were not expressed after infection with the capsule-deficient MC58 siaD mutant strain (Fig. 3). This represents 49.4% and 45%, respectively, of the total number of altered genes and suggests that half of the transcriptional responses of HBMEC are capsule dependent. These genes dependent on capsule expression could be assigned to all functional categories mentioned in Table 1. Table 4 shows the effects of the loss of capsule expression on a selection of genes which participate in the immune response. The capsule-deficient mutant induced more transcript than the WT N. meningitidis strain for genes, which are implicated in promoting a proinflammatory response (raw signal intensities higher for the MC58 siaD mutant than for the MC58 WT) (see the supplemental material). These patterns were further verified by FACS analysis for ICAM-1 expression (Fig. 1) and ELISA for IL-6 and TNF-α release (Fig. 2).

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FIG. 3.

Schematic representation of the number of regulated HBMEC genes induced following infection with the MC58 and MC58 siaD strains at 4 h postinfection (A) and 8 h postinfection (B). Tab., Table.

A total of 638 (at 4 h p.i.) and 544 (at 8 h p.i.) genes were expressed in response to both strains. A description of these genes is presented in Table 3, including apoptosis-related genes and genes encoding cytoskeleton/motility proteins, ILK, urokinase plasminogen activator (uPA), and hypoxia-inducible factor 1-responsive RTP801.

Apoptosis-associated proteins.Numerous HBMEC genes induced after meningococcal infection affected apoptosis. A number of key proapoptotic genes were up-regulated at 4 h p.i., including members of the Bcl2 family (bak and bad) and genes for calpain (MC58 siaD mutant at 4 h p.i. and 8 h p.i.), Fas-activated serine/threonine kinase, and apoptosis-specific protein, while bax and the death-associated protein 3 gene were down-regulated (Table 3). At the same time, several antiapoptotic genes were down-regulated, bclX, dad1, and the adenosine A1 receptor and Akt1 genes, while immediate-early response gene 1 was up-regulated. The regulation of TNF receptor-associated factor 1, which belongs to the “inhibitor of apoptosis” family, was time dependent, being down-regulated at 4 h p.i. and strongly up-regulated at 8 h p.i.

Infection-induced effects on host cell death.Since array data revealed up-regulation of proapoptotic genes and at the same time down-regulation of antiapoptotic genes in HBMEC, we next determined whether incubation with N. meningitidis leads to apoptosis in cultured HBMEC. There are conflicting reports in the literature regarding apoptosis induction by N. meningitidis (8, 33, 34, 39, 40). We selected different assays that cover a range of apoptotic events in human cells. In order to confirm the array data, real-time PCR was used to quantify gene expression of representative genes in MC58-infected HBMEC. The method described by Pfaffl (46) was used to calculate the changes in target gene expression relative to those in housekeeping gene expression (β-actin) (Table 4) . All genes that showed at least twofold regulated expression, as seen in the arrays compared to the controls, were regulated (except for apoptosis-specific protein up-regulation at 4 h p.i. after MC58 exposition), confirming the up-regulation of those genes already found to be regulated in the array analysis. We next monitored phosphatidylserine translocation on the cell surface by using annexin V staining and FACS analysis. After 8 h of infection, HBMEC exhibited no changes in annexin V staining (Fig. 4A). However, after 24 h of incubation, about 12% of cells were annexin V positive in response to MC58 (Fig. 4B). Infection with the unencapsulated MC58 siaD mutant revealed about 4% annexin V-positive cells.

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FIG. 4.

Annexin V-biotin binding by N. meningitidis-infected HBMEC. (A) Percentage of annexin V-biotin-positive cells in HBMEC culture, which was left untreated (1) or which was infected with strain MC58 (2) or the capsule-deficient MC58 siaD mutant (3) for 8 and 24 h (MOI, 10). After infection, cells were trypsinized, stained with annexin V-biotin, and detected using streptavidin-phycoerythrin conjugate by flow cytometry. Data represent means ± SEM for four independent experiments. *, P < 0.05 compared with the respective control; **, P < 0.01 compared with the respective control. (B) Representative dot plots of untreated HBMEC or cells infected with MC58 and the MC58 siaD mutant strain for 24 h (MOI, 10). Controls of specificity were used. Bacterial samples were incubated with annexin V-biotin. HBMEC infected with the MC58 WT were measured without primary annexin V-biotin staining. FL2-H, forward scattering intensity; SSC-H, side scattering intensity.

Infection of HBMEC with N. meningitidis leads to caspase 3 and AMPK-α activation and Akt kinase inhibition.Further experiments were carried out to determine whether infection of HBMEC with N. meningitidis leads to apoptosis in HBMEC. Assays for caspase 3 and AMPK-α activation were carried out by Western blot analysis. We decided to measure caspase 3 activation as an indicator of apoptosis induction since different upstream pathways leading to apoptosis depend on caspase 3 induction for final apoptotic execution. The kinetics of protein activation was assessed using antibodies that specifically recognized the cleaved caspase 3 (p17 and p19 subunits) and the phosphorylated forms of AMPK-α. Cell lysates prepared from uninfected HBMEC, cultured in the presence of human serum, were used as a control. As shown in Fig. 5, the unencapsulated N. meningitidis MC58 siaD strain induced cleavage of caspase 3 at 8 h of infection, with an increase of the large 17-kDa cleaved fragment after 24 h of infection. Furthermore, the unencapsulated mutant strain induced more cleaved 17-kDa fragment than the encapsulated parent strain (2.5- and 2.0-fold higher, as estimated by densitometry evaluation) (Fig. 5B).

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FIG. 5.

Effect of N. meningitidis infection on caspase 3, AMPK-α, and Akt activities in HBMEC. (A) HBMEC (10⁵ ml⁻¹) were incubated with the MC58 and MC58 siaD strains (10⁶ CFU ml⁻¹) at 37°C for the time indicated. Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis, and Western blotting was performed using antibodies specific for the phosphorylated and nonphosphorylated forms of the proteins of interest, as described in Materials and Methods. The results are representative of four identical experiments with (1) uninfected cells (control), (2) MC58-infected HBMEC, and (3) MC58 siaD strain-infected HBMEC. (B) The amounts of cleaved caspase 3, phosphorylated AMPK-α, and Akt were estimated by an NIH image analyzer program, and the mean increases (n-fold) or percentages of inhibition ± SEM were calculated. *, P < 0.05 relative to the respective control; **, P < 0.01 relative to the respective control; #, P < 0.05 relative to uninfected cells at 8 h of infection;”, P < 0.01 compared with controls.

Since AMPK was demonstrated to be able to participate in apoptosis regulation (4, 28), we next investigated the effects of meningococci on AMPK activity in HBMEC. AMPK has been described to be activated during cellular and environmental stress and regulates energy and metabolic homeostasis of the cell (22, 37). AMPK-α phosphorylation was detected after 24 h of infection in response to the MC58 and MC58 siaD strains (Fig. 5A). Wild-type and capsule-deficient MC58 bacteria led to almost identical increases of AMPK-α activity compared to uninfected cells (Fig. 5B). Moreover, expression analyses detected that the mRNAs of protein kinase B or Akt (PKB/Akt) showed a decrease in infected cells (Table 2). The serine-threonine protein kinase Akt is a multifunctional regulator of cell growth and glucose metabolism and is crucial to endothelial cell survival (13, 19). PKB/Akt either directly phosphorylates transcription factors that control the expression of pro- and antiapoptotic genes or promotes survival by directly phosphorylating key regulators of the apoptotic cascade (e.g., Bad) (44). In order to determine whether infection of HBMEC with meningococci not only decreased gene expression but also changed Akt activity, Western blot analyses were performed. After 8 h of infection, there was no visible effect on Akt activity (Fig. 5A). However, after 24 h of infection, we detected a marked inhibition of Akt phosphorylation in HBMEC infected with the MC58 and MC58 siaD strains by about 76.0% and 76.2%, respectively.