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Previous Article | Next Article 
Infection and Immunity, June 2008, p. 2685-2695, Vol. 76, No. 6
0019-9567/08/$08.00+0 doi:10.1128/IAI.01625-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Departments of Clinical Chemistry,1 Pediatrics, Ulleval University Hospital,2 Departments of Bacteriology and Immunology, The Norwegian Institute of Public Health,3 Faculty of Medicine, University of Oslo, Oslo, Norway4
Received 7 December 2007/ Returned for modification 25 January 2008/ Accepted 17 March 2008
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The interactions between N. meningitidis LPS and host cells have previously been studied in detail. Optimal cell activation requires hexa-acylated lipid A, phosphate head groups, and 2-keto-3-deoxyoctulosonic acid molecules in the LPS molecule (42, 48, 54). To exert their effects, the LPS molecules are translocated from the outer membrane of meningococci to the LPS-binding proteins, which function as a lipid shuttle and transport LPS to the membrane-bound or soluble CD14 (3, 35). In addition, myeloid differentiation protein 2, possibly modulating the lipid A structure, and TLR4 are essential components of the LPS receptor complex (2, 52). The intracellular signaling is conveyed via MyD88-dependent and -independent pathways resulting in activating of multiple gene-regulating components (55).
In previous studies we have used purified human monocytes as targets to try to dissect various pathophysiological mechanisms which are activated during meningococcal disease (2, 9, 29). Given the fact that N. meningitidis LPS is a major but not the only outer membrane molecule that may activate host cells, we have aimed to study the specificity of N. meningitidis LPS versus non-LPS molecules in the outer membrane of meningococci as they react with normal human monocytes (9). We have used microarray analysis to elucidate the specific effects of the LPS molecule by investigating the differences in global gene expression patterns after exposing monocytes to wild-type N. meningitidis (reference strain H44/76), LPS-deficient N. meningitidis (the N. meningitidis lpxA mutant), and purified N. meningitidis LPS. The results presented in this paper focus mainly on the effects of LPS presence by comparing the wild-type N. meningitidis and the LPS-deficient N. meningitidis in regard to both gene expression changes and proteins secreted to the culture medium. In addition, to substantiate the findings on LPS-induced transcriptional activation in human monocytes, we exploited the ability to quantify selected proteins in native biological systems, namely, in plasma from patients with meningococcal disease.
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Pooled human normal plasma. Heparinized whole blood was collected from consenting, healthy donors (n = 10) and immediately centrifuged (1,400 x g, 10 min, 20°C), and the plasma was pipetted off, mixed, aliquoted, and stored frozen at –70°C.
Acid-treated fetal calf serum (ATFCS) was prepared by acid treatment (to inactivate inhibitory serum-proteases and complement) of FCS tested to contain <1 EU/ml of LPS (51).
Wild-type N. meningitidis and LPS-deficient N. meningitidis (lpxA mutant). N. meningitidis strain H44/76, serogroup B, was isolated from a culture of blood from a Norwegian patient with fulminant septicemia. The strain belonged to the MLST32/ET-5 clone and was serologically characterized with monoclonal antibodies as B:15:P1.7,16 with the immunotype L3,7,9. The LPS-deficient N. meningitidis (lpxA mutant) was obtained by insertional inactivation of the lpxA gene in Neisseria meningitidis strain H44/76, serogroup B, kindly provided by Liana Steeghs and Peter van der Ley, National Institute of Public Health and Environment, Bilthoven, The Netherlands to the National Institute of Public Health, Oslo, Norway (40). The lpxA gene is required for adding the O-linked 3-OH fatty acid to UDP-N-acetylglucosamine, which is the first committed step in the lipid A biosynthesis pathway (40). The completely LPS-deficient (lpxA) mutant of N. meningitidis still expresses the immunodominant outer membrane proteins in normal amounts (47). The meningococci were grown overnight on chocolate agar base (GC) medium with IsoVitaleX (Becton Dickinson) and harvested into Hanks' balanced salt solution (HBSS) containing 0.1% (wt/vol) bovine serum albumin (BSA). The bacterial suspensions were heat inactivated at 56°C for 30 min. The number of bacteria was determined by quantitative real-time PCR (q-PCR) (see below). The amounts of LPS in wild-type N. meningitidis and the lpxA mutant, quantified using the LAL assay (5), were 29 EU/ml in 106 wild-type organisms and <1 EU/ml in 106 and 108 organisms of the lpxA mutant. The amounts of protein (4) in wild-type N. meningitidis (2.2 x 1010 CFU) and the lpxA mutant (1.2 x 1010 CFU) were 20 and 32 mg/ml, respectively.
Purified N. meningitidis LPS. Meningococcal LPS was extracted and purified from N. meningitidis prototype strain H44/76 (B:15:p1.16:L,3,7,9) as previously described (7). One nanogram of N. meningitidis LPS is equivalent to 33 EU/ml. The final product contained <0.3% protein and no particular nucleic acid (7).
Monocytes and patient plasma specimens. Elutriated, cryopreserved, purified human monocytes (>95% purity) (22) from different consenting, healthy donors (n = 3) were used (Biobank material access number 908; Ulleval University Hospital, Oslo, Norway). The patients' samples (n = 14) were collected after informed consent was obtained from parents, relatives, or patients and in accordance with guidelines approved by the Regional Medical Ethics Committee of Health Region I in Norway (Biobank material access number 948; Ulleval University Hospital, Oslo, Norway). The blood was collected and centrifuged, and plasma was pipetted off and aliquoted as described in detail earlier (8). The patients were categorized as to clinical presentation as previously described (8, 32).
Quantification of the number of bacteria by qPCR (N. meningitidis DNA quantification). In order to incubate the monocytes with the same number of meningococci, PCR quantification of N. meningitidis genomes, including genomes from both live and dead bacteria, was performed. Ten microliters of solutions of heat-inactivated wild-type or lpxA mutant N. meningitidis were added to 190 µl of pooled normal plasma (heparinized) and subjected to robotized isolation of N. meningitidis DNA (MagNA Pure LC robot, DNA isolation kit part no 03 003 990 001 [Roche]). Quantification of N. meningitidis DNA was performed using q-PCR as previously described (32), except with the sequence-specific hybridization probes (TIB Molbiol, Berlin, Germany) 5'-AGGATACGAATGTGCAGCTGAC-FL and 5'-LC Red640-GTGGCAATGTAGTACGAACTGTTGC-PH (0.3 µmol per reaction) and the Light Cycler Fast Start DNA Master Hybridization Probes Mix (catalog no. 12239272001; Roche) as the detection system.
Monocyte target assay. Elutriation-purified cryopreserved human monocytes were thawed, resuspended in 5% (vol/vol) ATFCS-RPMI 1640 containing 2% (vol/vol) penicillin-streptomycin, and seeded in microtiter plates (7.5 x 105 cells suspended in 250 µl ATFCS-RPMI/well). ATFCS-RPMI (250 µl containing wild-type N. meningitidis [106], N. meningitidis LPS [1 ng], N. meningitidis lpxA mutant [106 and 108], or vehicle) and pooled normal human plasma (500 µl) was added to duplicate wells, and the plates were sealed off and incubated for 0 and 3 h (37°C, 5% CO2). Pilot studies of both time course (0, 3, and 6 h) and concentrations of wild-type N. meningitidis, the N. meningitidis lpxA mutant, and N. meningitidis LPS were performed to determine the optimal gene expression changes of selected cytokines. Each plate was then centrifuged (47 x g, 5 min, 15°C) and the supernatant gently removed, aliquoted, and stored at –70°C until assayed for released proteins by the Luminex system (see below). Subsequently, the cells were harvested and stored in 500 µl lysis buffer Trizol reagent (Invitrogen, Carlsbad CA) at –70°C until subjected to total RNA isolation (see below).
Viability of monocytes. Cell viability was analyzed using a FACS Vantage DiVa flow cytometer (BD, San Jose, CA) with 488 nm excitation of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), detected through 530/22 and 630/22 filters, respectively. Human monocytes were seeded (Costar low-adherence plates) and stimulated with wild-type N. meningitidis (106), N. meningitidis LPS (1 ng), the N. meningitidis lpxA mutant (106 and 108), or vehicle for 3 hours prior to analysis. The number of cells was counted (Advia 60 hematology system; Bayer, Tarrytown, NY) before and after incubation to secure the same number of cells throughout the procedure. The harvested cells were suspended in annexin V binding buffer (BD, San Diego, CA) and annexin V-FITC (1 µg/ml) (BD), which was used to quantify the percentage of cells undergoing apoptosis. The plasma membrane integrity was investigated with PI (Sigma Aldrich, Oslo, Norway) at a final concentration of 2 µg/ml. Ten thousand events were acquired for each sample when analyzed in a PI versus annexin V-FITC dot plot by the BD FACS DiVa software version 4.1.2. Measurements of pulse-processed width versus area of forward light scatter signals were used to exclude doublets and aggregates from the analyses.
Opsonophagocytic activity. For the opsonophagocytic assay, the heat-inactivated wild-type and lpxA mutant N. meningitidis strains were labeled with the fluorescein-based probe carboxyfluorescein diacetate succinimidyl ester. Briefly, 20 µl of bacteria (approximately 1010 copies/ml) were washed in 1 ml HBSS supplemented with 0.2% BSA and suspended in 100 µl HBSS-BSA, and 5 µl carboxyfluorescein diacetate succinimidyl ester (1 mg/ml in dimethyl sulfoxide) was added. The mixtures were incubated for 5 min at room temperature, washed twice with 2 ml HBSS-BSA, centrifuged, and suspended in 200 µl HBSS-BSA. Human monocytes (from three different donors) were seeded as described above for 3 h in the presence of FITC-labeled wild-type N. meningitidis and the N. meningitidis lpxA mutant in 50% human pooled plasma and 50% ATFCS-RPMI. Additionally, plasma passed through a protein G column (GE Healthcare, Oslo, Norway) to remove immunoglobulin G antibodies was used. The phagocytosis was stopped by placing the plates on an ice bath until analysis (1). Phagocytosis was determined by fluorescence microscopy and flow cytometry. A forward- versus side-scatter histogram was used to identify the monocytes, and the percentage of fluorescence-positive cells was recorded in a separate histogram as a measure of bacterial uptake. To discriminate between adherent and internalized bacteria, trypan blue (0.5%, vol/vol) was added to the phagocytosed cells; trypan blue will quenches the fluorescence of adherent bacteria, whereas the fluorescence of internalized bacteria will remain unchanged. Thus, the preparation was analyzed first without trypan blue and then in the presence of trypan blue.
RNA preparation. Total RNA was extracted using the Trizol method (Invitrogen, Carlsbad, CA) and the RNeasy MinElute cleanup kit (Qiagen catalog no. 74204) according to the manufacturer's instructions. The isolated total RNA was quantified (Nano Drop spectrophotometer; Saveen Werner AB) and quality controlled using the Agilent BioAnalyzer 2100 system and the RNA 6000 Nano assay, giving RNA integrity number values ranging from 8.8 to 9.7.
Affymetrix gene expression profiling. One hundred nanograms of total RNA, spiked with 100 ng of poly(A) controls (Poly A RNA control kit, part 900433; Affymetrix, Santa Clara, CA), was subjected to analysis with the two-cycle cDNA synthesis kit following the manufacturer's (Affymetrix) recommended protocol for gene expression analysis. Biotinylated and fragmented cRNA (15 µl) was hybridized to the Affymetrix HG U133 Plus 2.0 array, representing 47,000 transcripts for 38,500 well-characterized human genes and 26 different cDNA synthesis controls. The signal intensities were detected with the Hewlett-Packard gene array scanner 3000 7G (Hewlett-Packard, Palo Alto, CA). The results are expressed as fold changes (FC), i.e., ratios of mean signal values from monocytes incubated with wild-type N. meningitidis (106), N. meningitidis LPS (1 ng), or the N. meningitidis lpxA mutant (106 and 108) to those from monocytes incubated with vehicle.
Statistical analysis of gene expression profiling.
The 18 scanned images were processed using GCOS 1.4 (Affymetrix). The CEL files were imported into Array Assist software (v5.2.0; Iobion Informatics LLC, La Jolla, CA) and normalized using the PLIER (probe logarithmic intensity error) algorithm in Array Assist to calculate relative signal values for each probe set. In order to filter for low signal values, the MAS5 algorithm in Array Assist was used to create a data set of absolute calls, showing the number of present and absent calls for each probe set. The filtration was performed by eliminating probe sets containing 
16 absent calls across the data set, resulting in a reduction of probe sets from 47,000 to 28,085. Duplicate or triplicate probe sets for a single gene were removed from the final data set in order to obtain the exact number of regulated genes. For expression comparisons of different groups, profiles were compared using both paired and unpaired t tests without corrections. Gene lists were generated with the criteria of a P value of <0.05 and an FC of |2|. Unique and common genes from the different gene lists were identified by Venn diagrams in Array Assist. MATLAB R2007a was used to identify FC to the subsets from the entire gene list. In addition, Ingenuity Pathway analysis (Redwood City, CA) was used for classifying genes into biological functions and signaling pathways.
RT-PCR.
The differential gene expression data were validated for selected transcripts using the TaqMan gene expression assays and the Applied Biosystems Prism 7900 HT sequence detection system. Thirty nanograms of pooled (10 ng from each of the three donors) total RNA from human monocytes induced by vehicle, wild-type N. meningitidis (106), N. meningitidis LPS 1 ng, and the N. meningitidis lpxA mutant (106 and 108) was reverse transcribed using Omniscript (Qiagen Ltd., Crawley, United Kingdom) (33). cDNA (9 µl) (diluted 1:3 in H2O) and 1 µl of either CD14-Hs02621496-s1, TLR4-Hs00152939-m1, TLR7-Hs00152971-m1, STAT1-Hs00234829-m1, STAT2-Hs01013126, SOCS2-Hs00374416-m1, CXCL9-Hs00171065-m1, CXCL11-Hs_00171138_m1, NEXN-Hs__00332124-m1, BCL2A1-Hs00187845-m1, IFNB1-Hs01077958_s1, or GAPDH-Hs99999905-m1 were added to 10 µl TaqMan universal PCR master mix (Applied Biosystems, part 4304437). The relative changes of each transcript, using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as endogenous control, were calculated using the software SDS Enterprise Database version 2.1 and the 
CT method (21). The results are expressed as relative quantities ( 2 ), i.e., the amount of targets (wild-type N. meningitidis [106], N. meningitidis LPS [1 ng] and the N. meningitidis lpxA mutant [108]) in incubated monocytes normalized to an endogenous reference (GAPDH) and relative to monocytes incubated with vehicle.
Quantification of protein levels in supernatants.
Supernatants harvested from human monocytes incubated with vehicle, wild-type N. meningitidis (106), N. meningitidis LPS (1 ng), or the N. meningitidis lpxA mutant (106 or 108) were analyzed using a microsphere-based multiplexing bioassay system with Xmap technology (Luminex Corporation, Austin, TX). Interleukin-6 (IL-6), IL-8, gamma interferon (IFN-
), macrophage inflammatory protein 1β (MIP-1β), and tumor necrosis factor alpha (TNF-
) were analyzed using the Bio-Plex Human Cytokine 11-Plex assay (Bio-Rad Laboratories Inc., Hercules, CA; lot no. X5006DMAWN). CSF-2 (Bio-Rad catalog no. 171A1180) was analyzed separately, while CXCL10 (R&D Systems Europe, Ltd., Abingdon, United Kingdom; catalog no. LUB266) and CXCL11 (R&D Systems; catalog no. LUB672) were analyzed together. All supernatants from each experiments (n = 3) were run in parallel. The analyses were performed as described by the manufacturer, who reported interassay variations (percent coefficient of variation) of below 10% for all except CXCL11, which ranged from 17 to 19%.
Quantification of CXCL10 and CXCL11 in samples from patients with meningococcal disease. Heparinized plasma (15 U/ml of blood) collected at hospital admission from patients with fulminant septicemia (n = 10) and distinct meningitis (n = 4) were quantified for CXCL10 and CXCL11 using Xmap technology (see above). All patients had positive blood culture. Healthy humans (laboratory personnel; n = 10) were analyzed as controls.
Quantification of N. meningitidis LPS in plasma from patients with meningococcal disease. Quantification of N. meningitidis LPS in plasma was performed with the LAL assay as described previously. The detection limit was 0.04 EU/ml (5, 8).
Quantification of N. meningitidis DNA copies in plasma from patients with meningococcal disease. Quantification of N. meningitidis DNA was performed using q-PCR as previously described (32).
Statistical analysis. The patients' data are given as median and range. The differences between patient groups were calculated using Spearman's rank order correlation in SSPS 14.0 (SPSS Inc., Chicago, IL). The level of significance was set at a P value of <0.05.
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RESULTS AND DISCUSSION |
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Similar results have been obtained by studying phagocytosis of capsulated and capsule-deficient N. meningitidis strains in human monocytes (20, 25). One may speculate as to whether the presence of LPS in the outer membrane protects the meningococci when monocytes encounter the bacteria and an incipient phagocytic process evolves. The mechanisms behind this phagocytic process are largely unknown. It is not antibody mediated, as shown by control experiments using immunoglobulin G-depleted plasma (data not shown). However, the complement system is operable, and it is likely that the mannose-binding lectin or the alternative pathway may play a role. It has thus been shown that wild-type meningococci may resist binding of mannose-binding lectin due to the terminal sialic acid of the LPS molecule (18). Thus, possibly through such mechanisms, the LPS-deficient N. meningitidis may be less protected against phagocytosis than the wild-type N. meningitidis in our in vitro system. The capsular polysaccharide produced by the wild-type N. meningitidis may also be more abundant than that produced by the LPS-deficient N. meningitidis, which could influence the phagocytosis.
One of the most up-regulated genes (FC, 155) (Table 3) in the presence of LPS is that for nexilin (symbol, NEXN). This is an F-actin-associated protein involved in the rearrangement of the cytoskeleton, which is found highly expressed in heart and skeletal muscles and furthermore has been shown to be involved in actin cytoskeletal remodeling mediating cell migration and adhesion in HeLa cells (50). Exposing monocytes to 108 LPS-deficient N. meningitidis organisms did not lead to expression of this transcript at all. To the best of our knowledge, nexilin has not yet been described in human monocytes, and its functional role in connection to phagocytosis still remains unclear.
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We also observed that in human monocytes incubated with wild-type N. meningitidis (106), 603 differentially expressed genes related to cell death were induced, compared with 22 and 542 genes when monocytes were incubated with LPS-deficient N. meningitidis at 106and 108 organisms, respectively (Table 1). In line with this closer examination of the cell death data (Table 1), 380 common cell death genes for wild-type N. meningitidis (106) and LPS-deficient N. meningitidis (108) were found (data not shown). This indicates that a substantial number of genes were similarly regulated (r = 0.75). All together, 163 genes were unique for the LPS-deficient N. meningitidis (108), and furthermore, 5 of these were related to the "necrotic processes." To what extent this may be reflected in increased necrosis (PI positivity) remains to be settled.
Identification of genes that were particularly sensitive to LPS. To dissect the specific effects of LPS presence in the meningococci, we compared the gene expression patterns from wild-type N. meningitidis and the LPS-deficient N. meningitidis (108). This comparison disclosed 2,288 genes, which we suggest to be "particularly sensitive to LPS." Of these, 80% were also found to be differentially expressed by purified N. meningitidis LPS. IFN-β and CXCL11 were the most up-regulated (Table 3) FC, >|5|), thus indicating that the presence of LPS molecules, among other molecules, leads to involvement of the TLR signaling (Table 4) and type I IFN (Table 5) pathways. To further substantiate these findings, we have dissected these signaling pathways by using Ingenuity pathway analysis.
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, MIP-1β, and TNF- mRNAs (Table 6) are examples of inflammatory genes that were found to be up-regulated in our study using either wild-type N. meningitidis, purified N. meningitidis LPS, or LPS-deficient N. meningitidis, showing involvement of the MyD88-dependent pathway. Supporting evidence that these up-regulations at the mRNA levels reflected alterations in the protein abundance was obtained by quantifying the responding secreted proteins in the supernatants (Table 6). However, our findings indicate that LPS-deficient N. meningitidis (108) is less potent in inducing selected cytokines than wild-type N. meningitidis. The presence of LPS (wild-type N. meningitidis as well as N. meningitidis LPS) additionally resulted in up-regulation of IFN-β mRNA, which would indicate involvement of the MyD88-independent pathway. In line with this is the finding of Toshchakov et al., who showed that activation of TLR4 by Escherichia coli LPS in murine macrophages resulted in an early induction of IFN-β through the MyD88-independent pathway (43). IFN-β release upon LPS stimulation has also been observed in human monocyte-derived dendritic cells by Severa et al. (38). It should furthermore be pointed out that the presence of LPS was a prerequisite for the up-regulation of several other genes assigned to the TLR signaling pathways, such as those for EIF2AK2 (IFN-inducible RNA-dependent protein kinase), IKBKB, NFKB1A, TLR5, TLR7, and TOLLIP (Table 4). Of these, TLR7, the receptor for single-stranded RNA in the endosomes, has been observed expressed in plasmacytoid dendritic cells upon LPS exposure (11, 26). TLR2, however, which is known to recognize lipoproteins, peptidoglycans, and lipoteichoic acids (28), was found to be present at 3 h but was not regulated by either of the inducers in the present study. Furthermore, the expression of TLR9, a receptor known to have specificity for bacterial nucleic acids, was found to be absent in our study despite the presence of bacterial DNA. TLR9 is located in intracellular compartments (13, 27) and is found constitutively expressed in neutrophils and monocytes (31). However, TLR9 signaling has been shown to be lower with heat-inactivated N. meningitidis as opposed to live N. meningitidis in HEK293 cells (28). To what extent methodological differences may contribute to the different results should be further investigated.
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LPS-sensitive genes in the JAK-STAT signaling pathway (type I IFN signaling pathway). The most affected canonical pathway in monocytes incubated with wild-type N. meningitidis was the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway (P = 8.5 10–6). Activation through this pathway by ILs, growth factors, or IFNs upon ligation of specific cell surface receptors results in the synthesis of a large number of ILs (>50), growth factors, and hormones as well as a series of inhibitory proteins (SOCS, PIAS, and PTP proteins) (45) involved in cell migration, cell proliferation, apoptosis, and immune development (23). Specificity in the gene expression pattern is generated from almost 40 receptors through combinations of four JAK and seven STAT family members (30). In the present study, we have shown that the presence of LPS leads to differential expression of IFNB1, ISGF3G (IRF9), JAK2, STAT1, and STAT2 (Table 5), all of which are involved in the IFN signaling pathway. Thus, the highly increased up-regulation of IFN-β (FC, 146) in the presence of LPS may give rise to formation of a trimeric ISGF-3 complex produced from STAT1, STAT2, and ISGF3G (14). This complex will translocate to the nucleus and induce transcription (14). Furthermore, the list of transcripts of "particularly LPS-sensitive genes" (Table 3) includes among others CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, IFIT5, IFI44, and ISG15, all of which are assumed to be IFN-β-inducible genes (34). As for protein presence, we could confirm this for CXCL10 and CXCL11 (Table 6) in the monocyte supernatants stimulated with wild-type N. meningitidis and N. meningitidis LPS but not in those stimulated with LPS-deficient N. meningitidis (108). These results relate to the findings of Severa et al., who demonstrated release from monocyte-derived dendritic cells of IFN-β-stimulated genes such as IRF7 and CXCL10 (38). Furthermore, Coelho et al. also showed CXCL11 to be up-regulated by IFN-β during differentiation of human monocytes to osteoclasts (10). Our findings, based on the several highly up-regulated transcripts and the detection of released proteins (CXCL10 and CXCL11) into the monocyte supernatants, may indicate that IFN-β release upon LPS stimulation may subsequently activate the IFN signaling pathway.
Quantification of CXCL10 and CXCL11 in plasma from patients with meningococcal disease. To investigate whether our in vitro findings of increased synthesis of CXCL10 and CXCL11 might occur in an in vivo situation, possibly signifying new circulating biomarkers, samples from patients with meningococcal disease (sepsis as well as meningitis) with positive blood culture collected at hospital admission and samples from healthy individuals were quantified for CXCL10, CXCL11, and LPS (Fig. 1). The median level of CXCL10 in plasma samples from patients with fulminant septicemia was 2,120 pg/ml (range, 997 to 5,709 pg/ml), which was significantly higher (P = 0.007) than that in patients with distinct meningitis (median, 395 pg/ml; range, 239 to 1,023 pg/ml). Healthy donors had a median value of 80 pg/ml (range, 38 to 186 pg/ml) (Fig. 1). The median level of CXCL11 in patients with fulminant septicemia was 848 pg/ml (range, 359 to 1,704 pg/ml), while patients with distinct meningitis had a median level of 819 pg/ml (range, 450 to 1,083 pg/ml). Healthy donors had a median level of 518 pg/ml (range, 271 to 977 pg/ml). There were no significant differences in the CXCL11 levels between the groups.
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Taking our results together, we propose that when human monocytes encounter meningococci, the presence of LPS leads to an early expression of IFN-β, most likely through the TLR-MyD88-independent pathway. Subsequently IFN-β may activate the JAK/STAT signaling pathway via IFNAR to form a STAT1/STAT2/IFR9 trimeric ISGF-3 complex through the type I IFN signaling pathway. This complex will interact with the IFN-stimulated response elements in the nucleus, and a yet-unknown number of IFN-β-inducible genes may be transcribed. Based on our experiments comparing gene expression patterns for wild-type N. meningitidis and LPS-deficient N. meningitidis (108), this implies that "particularly LPS-sensitive genes" are found within both the TLR and the JAK/STAT signaling pathways, influencing several biological processes such as chemotaxis, cell motility, and immune responses as evidenced by appearance of CXCL10 and CXCL11. In addition to these LPS-inducible pathways, other pathways are activated by other bacterial components. Possibly such in vitro-gained knowledge related to "the particularly LPS-sensitive genes" may lead to further investigations as to the presence of new biomarkers in meningococcal disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 24 March 2008.
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REFERENCES |
|---|
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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| 1. | Aase, A., L. M. Naess, R. H. Sandin, T. K. Herstad, F. Oftung, J. Holst, I. L. Haugen, E. A. Hoiby, and T. E. Michaelsen. 2003. Comparison of functional immune responses in humans after intranasal and intramuscular immunisations with outer membrane vesicle vaccines against group B meningococcal disease. Vaccine 21:2042-2051.[CrossRef][Medline] |
| 2. | Bjerre, A., B. Brusletto, R. Ovstebo, G. B. Joo, P. Kierulf, and P. Brandtzaeg. 2003. Identification of meningococcal LPS as a major monocyte activator in IL-10 depleted shock plasmas and CSF by blocking the CD14-TLR4 receptor complex. J. Endotoxin Res. 9:155-163.[Medline] |
| 3. | Bjerre, A., B. Brusletto, E. Rosenqvist, E. Namork, P. Kierulf, R. Ovstebo, G. B. Joo, and P. Brandtzaeg. 2000. Cellular activating properties and morphology of membrane-bound and purified meningococcal lipopolysaccharide. J. Endotoxin Res. 6:437-445.[CrossRef][Medline] |
| 4. | Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline] |
| 5. | Brandtzaeg, P., R. Øvstebø, and P. Kierulf. 2001. Quantitative detection of bacterial lipopolysaccharides in clinical specimens, p. 427-439. In A. J. Pollard and M. C. J. Maiden (ed.), Methods in molecular medicine. Meningococcal disease. The Humana Press, Totowa, NJ. |
| 6. | Brandtzaeg, P., A. Bjerre, R. Ovstebo, B. Brusletto, G. B. Joo, and P. Kierulf. 2001. Neisseria meningitidis lipopolysaccharides in human pathology. J. Endotoxin Res. 7:401-420.[CrossRef][Medline] |
| 7. | Brandtzaeg, P., K. Bryn, P. Kierulf, R. Ovstebo, E. Namork, B. Aase, and E. Jantzen. 1992. Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass-spectrometry, ultracentrifugation, and electron microscopy. J. Clin. Investig. 89:816-823.[Medline] |
| 8. | Brandtzaeg, P., P. Kierulf, P. Gaustad, A. Skulberg, J. N. Bruun, S. Halvorsen, and E. Sorensen. 1989. Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J. Infect. Dis. 159:195-204.[Medline] |
| 9. | Brandtzaeg, P., L. Osnes, R. Ovstebo, G. B. Joo, A. B. Westvik, and P. Kierulf. 1996. Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes. J. Exp. Med. 184:51-60. |
| 10. | Coelho, L. F., A. G. Magno de Freitas, F. J. Mennechet, A. Blangy, and G. Uze. 2005. Interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine CXCL11 expression. Proc. Natl. Acad. Sci. USA 102:11917-11922. |
| 11. | Fitzgerald-Bocarsly, P., and D. Feng. 2007. The role of type I interferon production by dendritic cells in host defense. Biochimie. 89:843-855.[Medline] |
| 12. | Gao, L. Y., and Y. A. Kwaik. 2000. The modulation of host cell apoptosis by intracellular bacterial pathogens. Trends Microbiol. 8:306-313.[CrossRef][Medline] |
| 13. | Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.[CrossRef][Medline] |
| 14. | Honda, K., and T. Taniguchi. 2006. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6:644-658.[CrossRef][Medline] |
| 15. | Humphries, H. E., M. Triantafilou, B. L. Makepeace, J. E. Heckels, K. Triantafilou, and M. Christodoulides. 2005. Activation of human meningeal cells is modulated by lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis and is independent of Toll-like receptor (TLR)4 and TLR2 signalling. Cell. Microbiol. 7:415-430.[CrossRef][Medline] |
| 16. | Ingalls, R. R., E. Lien, and D. T. Golenbock. 2001. Membrane-associated proteins of a lipopolysaccharide-deficient mutant of Neisseria meningitidis activate the inflammatory response through Toll-like receptor 2. Infect. Immun. 69:2230-2236. |
| 17. | Ingalls, R. R., E. Lien, and D. T. Golenbock. 2000. Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis. J. Endotoxin Res. 6:411-415.[CrossRef][Medline] |
| 18. | Jack, D. L., N. J. Klein, and M. W. Turner. 2001. Mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis. Immunol. Rev. 180:86-99.[CrossRef][Medline] |
| 19. | Kawai, T., and S. Akira. 2007. Antiviral signaling through pattern recognition receptors. J. Biochem. (Tokyo) 141:137-145. |
| 20. | Kolb-Maurer, A., A. Unkmeir, U. Kammerer, C. Hubner, T. Leimbach, A. Stade, E. Kampgen, M. Frosch, and G. Dietrich. 2001. Interaction of Neisseria meningitidis with human dendritic cells. Infect. Immun. 69:6912-6922. |
| 21. | Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods 25:402-408.[CrossRef][Medline] |
| 22. | Lund, P. K., G. B. Joo, A. B. Westvik, R. Ovstebo, and P. Kierulf. 2000. Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience. Scand. J. Clin. Lab. Investig. 60:357-365.[CrossRef][Medline] |
| 23. | Maher, S. G., A. L. Romero-Weaver, A. J. Scarzello, and A. M. Gamero. 2007. Interferon: cellular executioner or white knight? Curr. Med. Chem. 14:1279-1289.[CrossRef][Medline] |
| 24. | Massari, P., C. A. King, A. Y. Ho, and L. M. Wetzler. 2003. Neisserial PorB is translocated to the mitochondria of HeLa cells infected with Neisseria meningitidis and protects cells from apoptosis. Cell. Microbiol. 5:99-109.[CrossRef][Medline] |
| 25. | McNeil, G., M. Virji, and E. R. Moxon. 1994. Interactions of Neisseria meningitidis with human monocytes. Microb. Pathog. 16:153-163.[CrossRef][Medline] |
| 26. | Miggin, S. M. and L. A. O'Neill. 2006. New insights into the regulation of TLR signaling. J. Leukoc. Biol. 80:220-226. |
| 27. | Miyake, K. 2006. Roles for accessory molecules in microbial recognition by Toll-like receptors. J. Endotoxin Res. 12:195-204.[CrossRef][Medline] |
| 28. | Mogensen, T. H., S. R. Paludan, M. Kilian, and L. Ostergaard. 2006. Live Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis activate the inflammatory response through Toll-like receptors 2, 4, and 9 in species-specific patterns. J. Leukoc. Biol. 80:267-277. |
| 29. | Moller, A. S., R. Ovstebo, A. B. Westvik, G. B. Joo, K. B. Haug, and P. Kierulf. 2003. Effects of bacterial cell wall components (PAMPs) on the expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and the chemokine receptor CCR2 by purified human blood monocytes. J. Endotoxin Res. 9:349-360.[CrossRef][Medline] |
| 30. | Murray, P. J. 2007. The JAK-STAT signaling pathway: input and output integration. J. Immunol. 178:2623-2629. |
| 31. | O'Mahony, D. S., U. Pham, R. Iyer, T. R. Hawn, and W. C. Liles. 2008. Differential constitutive and cytokine-modulated expression of human Toll-like receptors in primary neutrophils, monocytes, and macrophages. Int. J. Med. Sci. 5:1-8.[Medline] |
| 32. | Ovstebo, R., P. Brandtzaeg, B. Brusletto, K. B. Haug, K. Lande, E. A. Hoiby, and P. Kierulf. 2004. Use of robotized DNA isolation and real-time PCR to quantify and identify close correlation between levels of Neisseria meningitidis DNA and lipopolysaccharides in plasma and cerebrospinal fluid from patients with systemic meningococcal disease. J. Clin. Microbiol. 42:2980-2987. |
| 33. | Ovstebo, R., K. B. Haug, K. Lande, and P. Kierulf. 2003. PCR-based calibration curves for studies of quantitative gene expression in human monocytes: development and evaluation. Clin. Chem. 49:425-432. |
| 34. | Paun, A., and P. M. Pitha. 2007. The IRF family, revisited. Biochimie 89:744-753.[Medline] |
| 35. | Post, D. M., D. Zhang, J. S. Eastvold, A. Teghanemt, B. W. Gibson, and J. P. Weiss. 2005. Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B. J. Biol. Chem. 280:38383-38394. |
| 36. | Pridmore, A. C., D. H. Wyllie, F. Abdillahi, L. Steeghs, P. van der Ley, S. K. Dower, and R. C. Read. 2001. A lipopolysaccharide-deficient mutant of Neisseria meningitidis elicits attenuated cytokine release by human macrophages and signals via toll-like receptor (TLR) 2 but not via TLR4/MD2. J. Infect. Dis. 183:89-96.[CrossRef][Medline] |
| 37. | Schubert-Unkmeir, A., O. Sokolova, U. Panzner, M. Eigenthaler, and M. Frosch. 2007. Gene expression pattern in human brain endothelial cells in response to Neisseria meningitidis. Infect. Immun. 75:899-914. |
| 38. | Severa, M., M. E. Remoli, E. Giacomini, J. Ragimbeau, R. Lande, G. Uze, S. Pellegrini, and E. M. Coccia. 2006. Differential responsiveness to IFN-alpha and IFN-beta of human mature DC through modulation of IFNAR expression. J. Leukoc. Biol. 79:1286-1294. |
| 39. | Sprong, T., N. Stikkelbroeck, P. van der Ley, L. Steeghs, L. van Alphen, N. Klein, M. G. Netea, J. W. van der Meer, and M. Van Deuren. 2001. Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response. J. Leukoc. Biol. 70:283-288. |
| 40. | Steeghs, L., R. den Hartog, A. den Boer, B. Zomer, P. Roholl, and P. van der Ley. 1998. Meningitis bacterium is viable without endotoxin. Nature 392:449-450.[CrossRef][Medline] |
| 41. | Stephens, D. S., B. Greenwood, and P. Brandtzaeg. 2007. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 369:2196-2210.[CrossRef][Medline] |
| 42. | Teghanemt, A., D. Zhang, E. N. Levis, J. P. Weiss, and T. L. Gioannini. 2005. Molecular basis of reduced potency of underacylated endotoxins. J. Immunol. 175:4669-4676. |
| 43. | Toshchakov, V., B. W. Jones, A. Lentschat, A. Silva, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, and S. N. Vogel. 2003. TLR2 and TLR4 agonists stimulate unique repertoires of host resistance genes in murine macrophages: interferon-beta-dependent signaling in TLR4-mediated responses. J. Endotoxin Res. 9:169-175.[Medline] |
| 44. | Uematsu, S., and S. Akira. 2006. Toll-like receptors and innate immunity. J. Mol. Med. 84:712-725.[CrossRef][Medline] |
| 45. | Uematsu, S., and S. Akira. 2007. Toll-like receptors and type I interferons. J. Biol. Chem. 282:15319-15323. |
| 46. | Uronen, H., A. J. Williams, G. Dixon, S. R. Andersen, P. van der Ley, M. van Deuren, R. E. Callard, and N. Klein. 2000. Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS). Clin. Exp. Immunol. 122:312-315.[CrossRef][Medline] |
| 47. | van der Ley, P., L. Steeghs, H. J. Hamstra, J. ten Hove, B. Zomer, and L. van Alphen. 2001. Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity. Infect. Immun. 69:5981-5990. |
| 48. | van der Ley, P., and L. Steeghs. 2003. Lessons from an LPS-deficient Neisseria meningitidis mutant. J. Endotoxin Res. 9:124-128.[Medline] |
| 49. | van Deuren, M., P. Brandtzaeg, and J. W. van der Meer. 2000. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin. Microbiol. Rev. 13:144-166. |
| 50. | Wang, W., W. Zhang, Y. Han, J. Chen, Y. Wang, Z. Zhang, and R. Hui. 2005. NELIN, a new F-actin associated protein, stimulates HeLa cell migration and adhesion. Biochem. Biophys. Res. Commun. 330:1127-1131.[CrossRef][Medline] |
| 51. | Wharton, S. A., and P. A. Riley. 1986. The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells. Cell Biochem. Funct. 4:189-195.[CrossRef][Medline] |
| 52. | Zimmer, S. M., S. M. Zughaier, Y. L. Tzeng, and D. S. Stephens. 2007. Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4. Glycobiology 17:847-856. |
| 53. | Zughaier, S., L. Steeghs, P. van der Ley, and D. S. Stephens. 2007. TLR4-dependent adjuvant activity of Neisseria meningitidis lipid A. Vaccine 25:4401-4409.[CrossRef][Medline] |
| 54. | Zughaier, S. M., Y. L. Tzeng, S. M. Zimmer, A. Datta, R. W. Carlson, and D. S. Stephens. 2004. Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway. Infect. Immun. 72:371-380. |
| 55. | Zughaier, S. M., S. M. Zimmer, A. Datta, R. W. Carlson, and D. S. Stephens. 2005. Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins. Infect. Immun. 73:2940-2950. |
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