Identification of Proteins Differentially Expressed in Human Monocytes Exposed to Porphyromonas gingivalis and Its Purified Components by Hi
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感染与免疫杂志 2006年第2期
Department of Periodontology and Oral Biology, School of Dental Medicine, Boston University, Boston, Massachusetts 02118
ABSTRACT
To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1 [MIP-1], and MIP-3) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.
INTRODUCTION
Periodontitis is a chronic infectious disease to which genetic, microbial, immunological, and environmental factors combine to influence disease risk and progression. Both bacterial virulence factors and host responses contribute to the connective tissue destruction and alveolar bone resorption characteristic of this disease (8, 13, 23). Porphyromonas gingivalis is a predominant periodontal pathogen which expresses a number of virulence factors involved in the pathogenesis of periodontitis. Among them, fimbriae play a critical role in mediating the bacterial interaction with host tissues, promoting bacterial adhesion to and invasion of the targeted sites (31). Fimbriae and LPS of this bacterium have been implicated in both the initiation and progression of disease. Previous studies have demonstrated that protein-free P. gingivalis LPS retains immunostimulatory activity in TLR4-deficient C3H/HeJ mice (37). Furthermore, it has been shown that, unlike enterobacterial LPS, P. gingivalis LPS and fimbriae use mostly TLR2 to induce innate immune responses in both human and mouse macrophages (32, 52).
In most cases of chronic inflammation triggered by an infection, a mononuclear cell infiltrate is typically present, and the prominent cell type found in mononuclear infiltrates is the monocyte. The mononuclear phagocyte plays an important role in regulation of the inflammatory host response, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. It has been demonstrated that monocytes constitute a substantial proportion of the cells recovered from the gingival tissues of patients with periodontitis (45). In addition, the numbers of monocytes in the inflammatory tissues of patients with periodontitis have been found to be higher than those from normal tissue (42). Overall, monocytes play a central role in orchestrating the response to gram-negative bacteria.
Understanding the molecular basis of the host response to bacterial infections is critical for preventing infection and also for minimizing the tissue damage resulting from an overly aggressive host response. The cellular and molecular events during the interaction of individual pathogenic components with host monocytes/macrophages have been characterized to some extent. Although LPS is generally considered to be a bacterial component that alerts the host to infection, P. gingivalis LPS is not as potent an activator of human monocytes as is Escherichia coli LPS, as measured by its relative activation of inflammation (1, 38). P. gingivalis LPS may selectively modify the host response as a means of facilitating colonization. Indeed, there have been reports suggesting a differential regulation of particular signaling pathways, such as the phosphatidylinositol-3-kinase-Akt pathway and the p38 MAPK pathway, in P. gingivalis LPS-induced production of proinflammatory and anti-inflammatory cytokines in human monocytes (5, 26). In addition, studies have indicated that P. gingivalis fimbriae activate human peripheral blood monocytes utilizing specific cellular receptors (32) and phosphorylated proteins (29), inhibit caspase-3-mediated apoptosis of monocytic cells (33), and induce monocyte adhesion to the endothelial cell surface and infiltration of monocytes into periodontal tissues of adult individuals with periodontitis (12). However, very little is known about the effect of live P. gingivalis bacteria relative to LPS and fimbriae on the human peripheral blood monocyte response.
Our previous study demonstrated qualitative and quantitative differences between the responses of monocytes to live P. gingivalis and to its fimbriae or LPS (52), supporting our hypothesis that unique signaling mechanisms are induced by live P. gingivalis. On this basis it is reasonable to suggest that a comprehensive understanding of host-bacterium interactions is currently incomplete, because most studies have examined the response to bacterial components rather than to live bacteria. To improve our understanding of the mechanisms by which human monocytes interact with live P. gingivalis, we attempted in the present study to identify proteins that are differentially modulated after treatment with live P. gingivalis relative to its purified LPS or fimbrial components. For this purpose, we employed the Becton-Dickinson PowerBlot Western Array screening system. This is a high-throughput Western blotting method which uses carefully formulated mixtures of subsets of 1,000 monoclonal antibodies to evaluate the differences in levels of cellular signaling proteins between total cell extracts from different cells or tissue. Treatments with live P. gingivalis, its LPS, or its FimA protein resulted in changes in the levels of several proteins, including transcription factors, signaling mediators, DNA synthesis and repair proteins, and proteins related to immune responses.
The P. gingivalis-, P. gingivalis LPS-, and P. gingivalis FimA-modulated proteins identified include some that have not been previously associated with the immune response. Our results provide additional support for the contention that P. gingivalis and its purified components modulate specific genes and proteins associated with the innate immune response, cell growth, and apoptosis.
MATERIALS AND METHODS
Abbreviations. The following terms used herein are abbreviated as indicated: lipopolysaccharides (LPS), Toll-like receptor 4 (TLR-4), mitogen-activated protein kinase (MAPK), sodium dodecyl sulfate (SDS), tumor necrosis factor alpha (TNF-), interleukin-6 (IL-6), enzyme-linked immunosorbent assay (ELISA), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), fibroblast growth factor 4 (FGF-4), insulin-like growth factor binding protein 2 (IFGBP-2), pulmonary and activated-regulated chemokine (PARC), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-related oncogene (GRO), tissue inhibitor of metalloproteinase 2 (TIMP-2), epithelial neutrophil-activating peptide 78 (ENA-78), neutrophil activating protein 2 (NAP-2), macrophage-derived chemokine (MDC), interferon-induced protein 10 (IP-10), regulated upon activation normal T-cell expressed and secreted (RANTES), oncostatin M (OSM), transforming growth factor 2 (TGF-2), IL enhancer-binding factor 2 (LIF3), signal transducer and activator of transcription factor 1 (STAT1), monocyte enhancer factor 2D (MEF2D), TNF superfamily member 6 (TNFSF6), culreticulin (CALR), double-stranded RNA-activated protein kinase (PRKR), clathrin heavy-chain polypeptide (CLTC), Ras-associated protein (RAB27A), and cell division cycle 42 (CDC42).
Bacterial strain and its components. P. gingivalis 381 (ATCC) was cultured in brain-heart infusion broth (Gibco) enriched with hemin (5 μg/ml) and menadione (1 μg/ml) in an anaerobic atmosphere (52). LPS from P. gingivalis 381 was extracted with phenol-water and purified by cesium chloride isopycnic density gradient ultracentrifugation followed by repurification, as we have previously described (52). The purity of LPS preparations was confirmed by amino acid analysis and by SDS-polyacrylamide gel electrophoresis with silver staining. FimA from P. gingivalis 381 was purified by size exclusion chromatography of sonicated extracts of P. gingivalis 381 (52). Fractions that were negative of any contaminating substances on silver-stained SDS-polyacrylamide gels were pooled, dialyzed, and lyophilized. The identity of the 41-kDa FimA protein was confirmed by N-terminal amino acid sequencing. The total FimA protein was quantified with the NanoOrange protein quantification kit (Molecular Probes, Eugene, OR).
Macrophage culture. Freshly elutriated human monocytes (>95% pure) were purchased from Advanced Biotechnologies (Columbia, MD). Monocytes were plated for 5 days to reach a density of 2 x 107 cells per 10 ml of Dulbecco modified Eagle medium (Invitrogen) with 20% fetal calf serum (Invitrogen), 10% human serum type AB (Nabi, Boca Raton, FL), and 50 μg/ml gentamicin (Invitrogen) in 10-cm-diameter tissue culture plates at 37°C in a humidified atmosphere containing 5% CO2 to enable differentiation into macrophages. On days 5 and 7, half of the medium was removed and replaced with medium lacking fetal calf serum. Medium on the cultured macrophages was changed to 5 ml of Dulbecco modified Eagle medium with 1% human serum on day 9, 1 h before experiments were begun.
Infection or treatment of macrophages with P. gingivalis or P. gingivalis components. Adherent macrophages were infected with live P. gingivalis bacteria at a ratio of 25:1 (bacteria to macrophages) at 37°C. Frozen stocks of P. gingivalis 381 were thawed, cultured, and diluted in medium to a concentration of 5 x 108 bacteria per 50 μl, to give multiplicities of infection (MOIs) of 25:1, and added to cultures of macrophages. Dilutions were plated on brain-heart infusion agar plates for anaerobic culturing, and colonies were counted to confirm the accuracy of dilution and viability of bacteria. In additional cultures, 10 μg/ml of purified LPS or FimA from P. gingivalis was added to medium containing 1% human serum and cells were incubated at 37°C. After incubation with P. gingivalis or its components, cells and supernatants were harvested at the indicated times.
Cytokine antibody array. The supernatants from P. gingivalis-infected and LPS- or FimA-stimulated macrophages derived from freshly elutriated human monocytes were analyzed with cytokine antibody arrays using RayBio Human Cytokine Antibody Array V (RayBiotech, Inc., Norcross, GA) according to the manufacturer's instructions. Briefly, cytokine array membranes were blocked in 2 ml of 1x blocking buffer for 30 min and then incubated with 1 ml of samples at room temperature for 1 to 2 h. Samples then were decanted from each container, and the membranes were washed three times with 2 ml of 1x wash buffer I, followed by two washes with 2 ml of 1x wash buffer II at room temperature with shaking. Membranes then were incubated in 1:250-diluted biotin-conjugated primary antibodies at room temperature for 1 to 2 h and washed as described above, before incubation in 1:1,000-diluted horseradish peroxidase-conjugated streptavidin for 30 to 60 min. Membranes were then washed thoroughly and exposed to a peroxidase substrate (detection buffers C and D; RayBiotech, Inc., Norcross, GA) for 5 min in the dark before imaging. Membranes were exposed to X-ray film (Kodak X-Omat AR) within 30 min ofaddition of the substrate. Signal intensities were quantified with Bio-Rad VersaDoc Imaging System 3000 and analyzed with Quantity One software (Bio-Rad). Biotin-conjugated immunoglobulin G served as a positive control at six spots, where it was used to identify membrane orientation and to normalize the results from different membranes that were being compared. For each spot, the net optical density level was determined by subtracting the background optical level from the total raw optical density level.
Sample preparation for PowerBlot and Western blot analysis. Cells were grown in 10-cm-diameter tissue culture plates and treated with live P. gingivalis, P. gingivalis LPS, or FimA for 24 h. Whole-cell lysates were prepared as follows: after removal of the medium by aspiration, cells were rinsed with phosphate-buffered saline three times. Boiling lysis buffer (10 mM Tris [pH 7.4], 1.0 mM sodium orthovanadate, and 1.0% SDS) was added to the plates to denature cellular proteins rapidly. The cell lysates were collected into a 50-ml conical tube and heated briefly in boiling water. The lysates were then sonicated for 10 s to shear genomic DNA. The protein concentration was determined with the Bio-Rad protein assay kit, and the samples were frozen at –80°C and sent on dry ice to BD Biosciences/Pharmingen (San Diego, CA) for PowerBlot analysis.
PowerBlot analysis. The levels of proteins were determined by PowerBlot analysis by BD Biosciences/Pharmingen (San Diego, CA). Briefly, samples containing 500 μg of protein in 500 μl of sample buffer were loaded in one large well on top of a 7.5 to 15% gradient SDS-polyacrylamide slab gel. Electrophoresis was conducted overnight at a constant current. The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). After transfer, the membranes were incubated in blocking buffer containing 5% nonfat dried milk. The membrane was clamped with a grid that isolates 40 lanes (chambers) oriented from the top to the bottom of the membrane. Mixtures containing four to five mouse monoclonal antibodies that recognize proteins of distinct molecular weights and antigenicities were added to each lane and allowed to hybridize for 1 h. A total of 995 antibodies were applied to seven templates per sample for this analysis. The blot was removed from the manifold, washed, and hybridized with goat anti-mouse antibody conjugated with horseradish peroxidase. The blots were developed with a chemiluminescence system (SuperSignal West Pico; Pierce, Rockford, IL). Each of the samples was analyzed on triplicate blots.
After exposure to X-ray films, the developed films were scanned, and the "trace" of each band was processed for densitometric analysis. Briefly, the trace includes a measure of both band intensity and band area. Standard average is the average trace for all bands on a blot. The individual band intensity was expressed as a percentage of the corresponding trace for average standards and for standards designated "percent lane average" to normalize for different exposures and protein loadings between gels. The percent lane average for bands of samples treated by P. gingivalis and its components (percent lane average for treated bands) was expressed as a percentage of the percent lane average for the corresponding control bands. The "percentage of control" was determined to express increases or decreases in protein expression. Changes are expressed as fold increase or decrease between the control percent lane average and the treated percent lane average. A summary file was provided, listing all protein expression changes detected in order of confidence, 1 through 5, with 5 being the highest level of confidence. The confidence level was based on fold change, reproducibility, and signal intensity (a good quality signal easily passes a visual inspection; it is hard for a low signal to pass a visual inspection). Levels are defined as follows: level 5, changes of >2-fold in triplicate from good quality signals; level 4, changes of 1.5- to 1.99-fold in triplicate from good quality signals; level 3, changes of 1.25- to 1.49-fold in triplicate from good quality signals; level 2, changes of >2-fold in triplicate from low signals; level 1, changes of >2-fold in duplicate from good quailty signals. In this article, we present only changes from levels 2 and higher.
Western blot analysis. Cell lysates prepared as described above or with cold lysis buffer (25 mM HEPES [pH 7.7], 400 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 0.5% Triton X-100, 3 mM dithiothreitol, 20 mM -glycerophosphate, 1mM sodium orthovanadate, and 25 mM para-nitrophenylphosphate and protease inhibitor mixture [Roche]) were analyzed by conventional Western blotting. Protein (50 μg) was electrophoresed through an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad) by electroblotting. The membranes were incubated in blocking buffer (5% nonfat dried milk, 10 mM Tris [pH 7.5], 100 mM NaCl, and 0.1% Tween 20). Immunoblotting for protein expression was performed with mouse monoclonal antibodies (BD Biosciences/Transduction Laboratories) against eight proteins selected from the proteins identified by the PowerBlot analysis. We used horseradish peroxidase-conjugated goat anti-mouse as a secondary antibody. The blots were developed with an enhanced chemiluminescence system (Amersham). Western blotting was repeated at least two times.
Cytokine ELISA. The supernatants from live P. gingivalis-, LPS-, or FimA-stimulated human macrophages were analyzed for TNF-, IL-6, and IL-10 by ELISA (R&D Systems, Minneapolis, MN).
Statistics. Statistical analysis was performed by the two-tailed Student t test for comparison between two groups. A P value of <0.05 was considered statistically significant. The correlation between two groups was quantified by use of the linear correlation coefficient.
RESULTS
Cytokine expression in human monocytes exposed to live P. gingivalis. To assess the impact of P. gingivalis on macrophage phenotype and function, freshly elutriated human monocytes were cultured as adherent monocyte-derived macrophages and exposed to live P. gingivalis. The cell culture supernatants were subjected to cytokine antibody array analysis (Fig. 1). In these isolated primary macrophage cultures, live P. gingivalis induced MCP-1, MIP-1, angiogenin, FGF-4, IGFBP-2, and PARC as early as 1 h after exposure (Fig. 1C). Then, GM-CSF, GRO, IL-6, IL-10, TNF-, MIP-3, and TIMP-2 were detected at 4 h, while ENA-78, GRO-, and IL-1 were detected at 24 h (Fig. 1D). In addition, among the earliest-induced cytokines, FGF-4 and PARC levels decreased or were not detected at 4 h, while MCP-1, MIP-1, MIP-3, and angiogenin levels decreased or were not detected at 24 h (Fig. 1C). NAP-2 and MDC were constitutively expressed in adherent monocyte-derived macrophages, but they were completely inhibited or degraded by live P. gingivalis after 24 h (Fig. 1B and C).
Differential effects of P. gingivalis and its purified components on the induction of cytokines in human monocytes. Following the initial infection, P. gingivalis can release its cell surface components such as LPS and FimA, which contribute to the progression of periodontitis. To establish whether P. gingivalis stimulated a profile of cytokine expression different from its LPS, compared to its FimA protein, in human monocytes, cultured monocyte-derived macrophages were treated with intact microorganisms, purified LPS, or purified FimA for 24 h and cytokine induction detected by cytokine array was compared with that of parallel unstimulated cell populations (Fig. 2). In these macrophage cultures, untreated controls were found constitutively to express IL-8, PARC, MCP-1, MDC, MIP-1, angiogenin, NAP-2, and TIMP-2 (Fig. 2A), while all of the other cytokines in the array were undetectable. Expression levels of IL-6, IL-10, TNF-, MIP-3, MIP-1, and ENA-78 in macrophages exposed to live P. gingivalis, purified LPS, and FimA were similar at 24 h, with IL-6, IL-10, TNF-, and MIP-3 being the dominant cytokines, whether triggered by P. gingivalis or by its components. A constitutively expressed cytokine, PARC, was not detectable either with P. gingivalis or with its two purified components after 24 h of exposure. However, expression of IL-1, GM-CSF, GRO-, and IGFBP-2 was induced only by live P. gingivalis, whereas, inversely, expression of IP-10, RANTES, MCP-1, MCP-2, MCP-3, OSM, and TGF-2 was induced only by its purified components LPS and FimA (Fig. 2B and C). MCP-1 was induced by P. gingivalis after a 4-h exposure but was down-regulated after 24 h of exposure (Fig. 1). Both P. gingivalis and its FimA protein induced I-309/CCL-1, while LPS induced IGFBP-3. TIMP-2 was up-regulated by live P. gingivalis but down-regulated by the purified LPS.
In order to check the reliability of the relative differences observed between cultures treated with P. gingivalis and with its components, we quantified the levels of TNF-, IL-6, and IL-10, three cytokines that showed large fold differences in levels among cultures. The actual TNF-, IL-6, and IL-10 levels in the cell supernatants used in the antibody arrays were determined by a conventional ELISA, as described in Materials and Methods. The actual TNF-, IL-6, and IL-10 levels (in pg/ml) showed the same patterns seen in the arrays (Table 1). The levels of TNF-, IL-6, and IL-10 detected by ELISA correlated well with the net optical intensity differences seen in the antibody array experiments. The linear correlation coefficients (r values) for TNF-, IL-6, and IL-10 were 0.9911, 0.9936, and 0.9962, respectively.
Identification of differentially expressed proteins induced by P. gingivalis or its purified components as revealed by PowerBlot analysis. Extracts of human monocyte-derived macrophages grown for 24 h in control medium or in media supplemented with live P. gingivalis, LPS, or FimA were subjected to PowerBlot analysis. A representative blot of proteins extracted from untreated cells is shown in Fig. 3A. Enlarged parts of this control blot compared with corresponding regions from blots derived from P. gingivalis-treated or cell surface component-treated cells demonstrating changes (increases or decreases) in the levels of several proteins (circled or enclosed within rectangles) are presented in Fig. 3B through J. The analysis of the PowerBlot antibody array data indicated that P. gingivalis and its purified components induced both increases and decreases in the levels of specific proteins. Thus, treatment of the human macrophages with live P. gingivalis, its LPS, or its FimA protein led to up-regulation of 12, 8, and 10 proteins and down-regulation of 15, 8, and 17 proteins, respectively (Table 2). Included in this table are proteins involved in cell signaling, cell adhesion, cell growth, gene transcription, DNA repair, apoptosis, and other fundamental cellular processes. The changes induced in protein levels by P. gingivalis and its purified LPS and FimA could be categorized into several types: (i) changes that were similar for the three stimuli, (ii) changes that were induced by two of the three stimuli, (iii) changes that were induced by only one of the stimuli, and (iv) changes that were modulated uniquely by each stimulus.
Validation of PowerBlot results by conventional Western blotting. To confirm the PowerBlot data, we selected eight proteins (ILF3, STAT1, MAPK3, MEF2D, TNFSF6, CALR, STAT6, and PRKR) with various levels of differential expression and representing different confidence levels of changes detected by PowerBlot for analysis by conventional Western blotting (Fig. 4). With one exception (LPS-induced MAPK3), the conventional Western blotting analysis confirmed the differential expression of these eight proteins, which are listed in Table 3. The amounts of MEF2D, CALR, and PRKR induced by LPS relative to controls increased 2.03-, 2.54-, and 3.64-fold, respectively (Fig. 4), concordant with their 2.62-, 2.98-, and 3.7-fold differential expressions observed in the PowerBlot analysis results (Table 2). The amounts of ILF3, TNFSF6, CALR, PRKR, STAT1, and MAPK3 induced by FimA relative to controls increased 1.53-, 1.68-, 5.62-, 1.87-, 0.64-, and 0.37-fold, respectively (Fig. 4), in good agreement with their 1.43-, 2.22-, 2.98-, and 5.59-fold increases and 3.35- and 2.31-fold decreases, respectively, observed in the PowerBlot analysis results (Table 2). The relative ratios of STAT1, MAPK3, STAT6, PRKR, and CALR to controls induced by P. gingivalis were 0.08, 0.43, 0.32, 0.66, and 4.21, respectively (Fig. 4); these are comparable with the 16.63-, 4.20-, and 3.46-fold decrease and the 6.15-fold increase in differential expression observed for these proteins, respectively, in the PowerBlot analysis. MAPK3 induced by LPS was the only exception that was observed: its levels decreased according to the Western blot analysis but were unchanged according to the PowerBlot analysis. P. gingivalis, its purified LPS, and FimA all increased CALR expression in human monocytes in the PowerBlot analysis. This was also confirmed by two-dimensional gel electrophoresis followed by mass spectrometry (data not shown).
DISCUSSION
The innate immune response to P. gingivalis is initiated when the microorganisms interact with cell surface pattern receptors, including TLRs, to signal and/or become internalized. Recognition occurs largely through the bacterial cell surface constituents, such as LPS and fimbriae, and those cell surface constituents bind to TLR2 and also may bind to the glycosylphosphatidylinositol-linked protein CD14 (9, 10, 48) to induce inflammatory responses. It is thought that many of the effects of bacteria on host immune and inflammatory responses are mediated by changes in gene transcription. Indeed, changes in gene expression have been identified in cells treated with P. gingivalis and its purified LPS or fimbriae using differential display (35) and cDNA array (15) techniques, both of which are focused on mRNA analysis. Changes at the mRNA level may not correspond to changes in protein levels or to changes in activity, especially when those depend on posttranslational modifications and/or protein stability. Therefore, it is important to investigate the effects of P. gingivalis and its components on protein levels. Thus far, few reports have addressed the effects of P. gingivalis and its components on the modulation of proteins in human peripheral monocytes using large-scale proteomics. We designed this study to compare the effects of P. gingivalis and its components to see whether they cause distinct biological effects in human peripheral monocyte-derived macrophages. This article provides the first description of host proteins by P. gingivalis and its components LPS and FimA.
Using cytokine antibody arrays, we first investigated cytokines, the major inflammatory mediators induced by live P. gingivalis. At the initial interaction with macrophages, P. gingivalis induced chemokines (e.g., MCP-1, MIP-1, and PARC) involved in macrophage and neutrophil recruitment and in the antigen-specific T helper cell type 1 immune response (14, 47). However, this immune response may be suppressed after 24 h of exposure to P. gingivalis, since those chemokines, along with DMC/CCL22 and NAP-2, were dramatically reduced or disappeared completely after that time (Fig. 1 and 2). Following the initial response, expression of an extensive set of cytokines (including TNF-, IL-6, and IL-1) and other mediators transiently escalated subsequent to infection. Simultaneously, increases in IL-10 and TGF- may have contributed to the dampening of the initial activation response (3, 4). TNF- and IL-1, proinflammatory cytokines that activate multiple signal transduction pathways to influence both immune and nonimmune cell functions, are known to inhibit macrophage apoptosis (24). This agrees with previous observations that infection with P. gingivalis appears to protect macrophages from apoptosis (30, 33, 36, 50), thereby maintaining the pool of infected and infectible targets.
The immune and inflammatory responses to live bacteria may not be triggered simply by bacterial cell surface components, although these are among the best-characterized pattern recognition receptor ligands. Our cytokine antibody array data show that LPS and FimA induce similar patterns of cytokine production by human monocyte-derived macrophages; however, the patterns of cytokines produced by human macrophages stimulated by LPS or FimA differed substantially from that observed when the cells were challenged with live P. gingivalis (Fig. 2). It seems that the host defense system senses live bacteria differently than individual bacterial constituents, and thus it launches a different immune response. Our data are consistent with previous studies demonstrating that both P. gingivalis LPS and FimA signal through a common pattern recognition receptor, TLR2, whereas live P. gingivalis signals through both TLR2 and TLR4 in the induction of cytokine production by mouse peritoneal macrophages (9, 52).
In order to identify intracellular signaling pathways activated during P. gingivalis infection, we identified intracellular proteins modulated in human macrophages after interaction with live P. gingivalis and its by-products, LPS and FimA, by using the PowerBlot array screening system. The analysis demonstrates differences in the levels of several proteins after macrophage exposure to P. gingivalis, its LPS, or its FimA protein (Table 2). These proteins included those associated with immune response, both Ser/Thr and Tyr protein kinases, heat shock response proteins, transcription factors, DNA repair enzymes, and proteins engaged in vesicular transport, apoptosis, cell cycle, and cell adhesion. Additional studies are required to determine whether some or any of the changes in the levels of proteins are due to transcriptional regulation via ligand-specific receptors and whether they are the cause or consequence of the biological effects. It is noteworthy that we have analyzed changes that occurred after 24 h of cell treatment, and so some of the changes may be earlier than changes in cell cycle or cell viability. Here we have elected to highlight several of the modulated proteins that may be related to the mechanisms underpinning the effects of P. gingivalis and its constituents on infection, inflammation, and immune response.
We observed that similar changes in CLTC, CALR, RAB27A, and MAPK3 levels were induced by P. gingivalis, its LPS, and its FimA protein, suggesting a common mechanism of action. CLTC, a clathrin heavy-chain protein, was down-regulated after exposure to all three treatments, which may result in attenuated macrophage chemotaxis and phagocytosis (18, 46). A Ca2+ binding protein, CALR, which was reported to modulate the host immune response by binding to complement C1q, thereby participating in the inflammatory processes associated with vascular or atherosclerotic lesions, autoimmune diseases, or infections (7), was up-regulated by these three stimuli. The GTP-binding protein RAB27A, which appears to be a key effector of cytotoxic granule exocytosis, a pathway essential for immune homeostasis (28), was also down-regulated under all three conditions. P. gingivalis, its LPS, and its FimA protein all repressed the basal expression of MAPK3 (ERK1) in macrophages; suppression of MAPK3 was reported to facilitate an adaptive regulation in the process of cell proliferation under cell growth arrest conditions (11). All of these functions may favor its establishment and propagation within the host after P. gingivalis infection or enhance the host's chronic inflammatory reactions.
Purified LPS from P. gingivalis specifically induced MEF2D, IFIT4, and CDC42. MEF2D is a member of the myocyte enhancer factor 2 family of transcription factors and has been shown to increase CD14 expression in monocytes/macrophages (34); up-regulation of the MEF2D protein by P. gingivalis LPS may enhance macrophage responses to LPS by possibly increasing CD14 expression. That LPS up-regulates the interferon-inducible antiviral protein IFIT4 was also found by other investigators (16); however, its role in biological processes and molecular function here is still unknown and needs further investigation. The GTPase CDC42 is a GTP-binding protein that is involved in macrophage phagocytosis via the calcium-promoted Ras inactivator (51). However, P. gingivalis LPS may not induce phagocytosis and oxidative burst by activating CDC42, because we did not detect the up-regulation of the calcium-promoted Ras inactivator in macrophages treated with P. gingivalis LPS.
FimA has been shown to up-regulate TNFSF6 (CD95L) and ILF3. A type II transmembrane protein of the TNF family of death factors, TNFSF6 has shown to be associated with the establishment of immune privilege and tumor survival (39). Interaction between TNFSF6 and its receptor can regulate key events in cellular activation, proliferation, differentiation, cell death, and survival of immune cells and other tissues (22). ILF3 is a transcription factor required for T-cell expression of IL-2 (25). ILF3 binds to a sequence in the IL-2 enhancer known as antigen receptor response element 2. In addition, ILF3 can bind RNA and is an essential component for encapsidation and protein priming of hepatitis B viral polymerase (17). That FimA induced TNFSF6 and ILF3 expression may indicate that it plays an important role in affecting the host immune system.
Live P. gingivalis dramatically down-regulated expression of the transcription factor STAT6. This is an intriguing finding, in light of other data that STAT6 deficiency confers enhanced immunity against tumors (44) as well as against parasite infection (40, 43). STAT6-deficient mice generate excess nitric oxide in their macrophages compared to the macrophages from wild-type mice, which produce arginase instead of nitric oxide (44). Nitric oxide-producing macrophages have a strong ability to induce antigen-specific CD4+ T-cell proliferation in response to unrelated antigens (41). It seems that during the acute infection stage (24 h after infection), P. gingivalis turns on nitric oxide production by macrophages as a host defense mechanism, accomplished by down-regulating the expression of STAT6.
The level of STAT1 was decreased in macrophages treated with either FimA or live P. gingivalis. STAT1 plays an essential role in the caspase-independent cell death of activated macrophages through the p38 MAPK/STAT1/reactive oxygen species pathway (20). Decreased STAT1 levels were reported to decrease superoxide production in macrophages (20). Down-regulation of STAT1 by P. gingivalis or by its FimA protein may be one of the several survival mechanisms used by P. gingivalis to evade host defenses.
STAT3 is a transcription factor mediating the anti-inflammatory properties of IL-10 (49). It has been reported that mice with a STAT3 deficiency display excessive local and systemic inflammation and fail to facilitate bacterial clearance (27). We noticed that P. gingivalis LPS down-regulated and FimA up-regulated STAT3 expression in macrophages; this may indicates that LPS plays a negative role while FimA plays a positive role in stimulation of anti-inflammatory activity and bacterial clearance during P. gingivalis infection. In addition, the STAT3 level in macrophages was not changed by whole bacterial treatment compared to that of untreated macrophages; it seems that the whole bacteria balanced the role of LPS and FimA on inducing STAT3 expression in macrophages.
PRKR, an interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (p68 kinase), acts as a tumor suppressor by actively participating in apoptosis (21). PRKR is also essential for antiviral activity by inhibiting both viral and cellular protein synthesis (19). Interestingly, it was increased only by LPS and decreased by both live P. gingivalis and FimA. The abilities of live P. gingivalis and FimA to decrease PRKR levels may be important for the antiapoptotic activities observed by other investigators (50). However, the ability of P. gingivalis LPS to increase PRKR levels may not related to apoptogenic activity, because P. gingivalis LPS also possesses antiapoptotic properties (30).
Another interesting observation of this study was the down-regulation of TPL-2 (MAP3K8) by live P. gingivalis and its LPS. P. gingivalis LPS is a TLR2 agonist; it was reported to inhibit a TLR4 agonist, E. coli LPS-induced TNF- production, with IB kinase activity being unperturbed (6). This TLR "heterotolerance" induced by P. gingivalis LPS may come about through down-regulation of TPL-2, via an IB kinase-independent pathway, because TPL-2 is essential for TLR4 activation of the ERK MAPK cascade in E. coli LPS-stimulated macrophages and subsequent up-regulation of genes involved in innate immune responses (2). The down-regulation of TPL-2 may also be associated with the down-regulation of MAPK3 detected in this study, which could weaken the innate host immune responses to bacterial infection.
In summary, the large-scale screening of proteins modulated after treatment of human monocyte-derived macrophages with live P. gingivalis, its LPS, and its FimA protein has enabled us to identify proteins that are implicated in the effects of P. gingivalis and its components on the innate immune responses of human monocytes and macrophages. P. gingivalis and its components appear to stimulate different protein expression patterns in macrophages that may reflect intrinsic functional differences in these stimuli. This differential modulation of proteins in host cells may well coordinate with their specific roles in triggering host inflammatory/immune responses during P. gingivalis infection.
ACKNOWLEDGMENTS
This work was supported by grant RO1 DE 15989 from NIDCR.
Present address: The Pulmonary Center, School of Medicine, Boston University Medical Center, 15 Stoughton Street R-304, Boston, MA 02118.
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ABSTRACT
To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1 [MIP-1], and MIP-3) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.
INTRODUCTION
Periodontitis is a chronic infectious disease to which genetic, microbial, immunological, and environmental factors combine to influence disease risk and progression. Both bacterial virulence factors and host responses contribute to the connective tissue destruction and alveolar bone resorption characteristic of this disease (8, 13, 23). Porphyromonas gingivalis is a predominant periodontal pathogen which expresses a number of virulence factors involved in the pathogenesis of periodontitis. Among them, fimbriae play a critical role in mediating the bacterial interaction with host tissues, promoting bacterial adhesion to and invasion of the targeted sites (31). Fimbriae and LPS of this bacterium have been implicated in both the initiation and progression of disease. Previous studies have demonstrated that protein-free P. gingivalis LPS retains immunostimulatory activity in TLR4-deficient C3H/HeJ mice (37). Furthermore, it has been shown that, unlike enterobacterial LPS, P. gingivalis LPS and fimbriae use mostly TLR2 to induce innate immune responses in both human and mouse macrophages (32, 52).
In most cases of chronic inflammation triggered by an infection, a mononuclear cell infiltrate is typically present, and the prominent cell type found in mononuclear infiltrates is the monocyte. The mononuclear phagocyte plays an important role in regulation of the inflammatory host response, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. It has been demonstrated that monocytes constitute a substantial proportion of the cells recovered from the gingival tissues of patients with periodontitis (45). In addition, the numbers of monocytes in the inflammatory tissues of patients with periodontitis have been found to be higher than those from normal tissue (42). Overall, monocytes play a central role in orchestrating the response to gram-negative bacteria.
Understanding the molecular basis of the host response to bacterial infections is critical for preventing infection and also for minimizing the tissue damage resulting from an overly aggressive host response. The cellular and molecular events during the interaction of individual pathogenic components with host monocytes/macrophages have been characterized to some extent. Although LPS is generally considered to be a bacterial component that alerts the host to infection, P. gingivalis LPS is not as potent an activator of human monocytes as is Escherichia coli LPS, as measured by its relative activation of inflammation (1, 38). P. gingivalis LPS may selectively modify the host response as a means of facilitating colonization. Indeed, there have been reports suggesting a differential regulation of particular signaling pathways, such as the phosphatidylinositol-3-kinase-Akt pathway and the p38 MAPK pathway, in P. gingivalis LPS-induced production of proinflammatory and anti-inflammatory cytokines in human monocytes (5, 26). In addition, studies have indicated that P. gingivalis fimbriae activate human peripheral blood monocytes utilizing specific cellular receptors (32) and phosphorylated proteins (29), inhibit caspase-3-mediated apoptosis of monocytic cells (33), and induce monocyte adhesion to the endothelial cell surface and infiltration of monocytes into periodontal tissues of adult individuals with periodontitis (12). However, very little is known about the effect of live P. gingivalis bacteria relative to LPS and fimbriae on the human peripheral blood monocyte response.
Our previous study demonstrated qualitative and quantitative differences between the responses of monocytes to live P. gingivalis and to its fimbriae or LPS (52), supporting our hypothesis that unique signaling mechanisms are induced by live P. gingivalis. On this basis it is reasonable to suggest that a comprehensive understanding of host-bacterium interactions is currently incomplete, because most studies have examined the response to bacterial components rather than to live bacteria. To improve our understanding of the mechanisms by which human monocytes interact with live P. gingivalis, we attempted in the present study to identify proteins that are differentially modulated after treatment with live P. gingivalis relative to its purified LPS or fimbrial components. For this purpose, we employed the Becton-Dickinson PowerBlot Western Array screening system. This is a high-throughput Western blotting method which uses carefully formulated mixtures of subsets of 1,000 monoclonal antibodies to evaluate the differences in levels of cellular signaling proteins between total cell extracts from different cells or tissue. Treatments with live P. gingivalis, its LPS, or its FimA protein resulted in changes in the levels of several proteins, including transcription factors, signaling mediators, DNA synthesis and repair proteins, and proteins related to immune responses.
The P. gingivalis-, P. gingivalis LPS-, and P. gingivalis FimA-modulated proteins identified include some that have not been previously associated with the immune response. Our results provide additional support for the contention that P. gingivalis and its purified components modulate specific genes and proteins associated with the innate immune response, cell growth, and apoptosis.
MATERIALS AND METHODS
Abbreviations. The following terms used herein are abbreviated as indicated: lipopolysaccharides (LPS), Toll-like receptor 4 (TLR-4), mitogen-activated protein kinase (MAPK), sodium dodecyl sulfate (SDS), tumor necrosis factor alpha (TNF-), interleukin-6 (IL-6), enzyme-linked immunosorbent assay (ELISA), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), fibroblast growth factor 4 (FGF-4), insulin-like growth factor binding protein 2 (IFGBP-2), pulmonary and activated-regulated chemokine (PARC), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-related oncogene (GRO), tissue inhibitor of metalloproteinase 2 (TIMP-2), epithelial neutrophil-activating peptide 78 (ENA-78), neutrophil activating protein 2 (NAP-2), macrophage-derived chemokine (MDC), interferon-induced protein 10 (IP-10), regulated upon activation normal T-cell expressed and secreted (RANTES), oncostatin M (OSM), transforming growth factor 2 (TGF-2), IL enhancer-binding factor 2 (LIF3), signal transducer and activator of transcription factor 1 (STAT1), monocyte enhancer factor 2D (MEF2D), TNF superfamily member 6 (TNFSF6), culreticulin (CALR), double-stranded RNA-activated protein kinase (PRKR), clathrin heavy-chain polypeptide (CLTC), Ras-associated protein (RAB27A), and cell division cycle 42 (CDC42).
Bacterial strain and its components. P. gingivalis 381 (ATCC) was cultured in brain-heart infusion broth (Gibco) enriched with hemin (5 μg/ml) and menadione (1 μg/ml) in an anaerobic atmosphere (52). LPS from P. gingivalis 381 was extracted with phenol-water and purified by cesium chloride isopycnic density gradient ultracentrifugation followed by repurification, as we have previously described (52). The purity of LPS preparations was confirmed by amino acid analysis and by SDS-polyacrylamide gel electrophoresis with silver staining. FimA from P. gingivalis 381 was purified by size exclusion chromatography of sonicated extracts of P. gingivalis 381 (52). Fractions that were negative of any contaminating substances on silver-stained SDS-polyacrylamide gels were pooled, dialyzed, and lyophilized. The identity of the 41-kDa FimA protein was confirmed by N-terminal amino acid sequencing. The total FimA protein was quantified with the NanoOrange protein quantification kit (Molecular Probes, Eugene, OR).
Macrophage culture. Freshly elutriated human monocytes (>95% pure) were purchased from Advanced Biotechnologies (Columbia, MD). Monocytes were plated for 5 days to reach a density of 2 x 107 cells per 10 ml of Dulbecco modified Eagle medium (Invitrogen) with 20% fetal calf serum (Invitrogen), 10% human serum type AB (Nabi, Boca Raton, FL), and 50 μg/ml gentamicin (Invitrogen) in 10-cm-diameter tissue culture plates at 37°C in a humidified atmosphere containing 5% CO2 to enable differentiation into macrophages. On days 5 and 7, half of the medium was removed and replaced with medium lacking fetal calf serum. Medium on the cultured macrophages was changed to 5 ml of Dulbecco modified Eagle medium with 1% human serum on day 9, 1 h before experiments were begun.
Infection or treatment of macrophages with P. gingivalis or P. gingivalis components. Adherent macrophages were infected with live P. gingivalis bacteria at a ratio of 25:1 (bacteria to macrophages) at 37°C. Frozen stocks of P. gingivalis 381 were thawed, cultured, and diluted in medium to a concentration of 5 x 108 bacteria per 50 μl, to give multiplicities of infection (MOIs) of 25:1, and added to cultures of macrophages. Dilutions were plated on brain-heart infusion agar plates for anaerobic culturing, and colonies were counted to confirm the accuracy of dilution and viability of bacteria. In additional cultures, 10 μg/ml of purified LPS or FimA from P. gingivalis was added to medium containing 1% human serum and cells were incubated at 37°C. After incubation with P. gingivalis or its components, cells and supernatants were harvested at the indicated times.
Cytokine antibody array. The supernatants from P. gingivalis-infected and LPS- or FimA-stimulated macrophages derived from freshly elutriated human monocytes were analyzed with cytokine antibody arrays using RayBio Human Cytokine Antibody Array V (RayBiotech, Inc., Norcross, GA) according to the manufacturer's instructions. Briefly, cytokine array membranes were blocked in 2 ml of 1x blocking buffer for 30 min and then incubated with 1 ml of samples at room temperature for 1 to 2 h. Samples then were decanted from each container, and the membranes were washed three times with 2 ml of 1x wash buffer I, followed by two washes with 2 ml of 1x wash buffer II at room temperature with shaking. Membranes then were incubated in 1:250-diluted biotin-conjugated primary antibodies at room temperature for 1 to 2 h and washed as described above, before incubation in 1:1,000-diluted horseradish peroxidase-conjugated streptavidin for 30 to 60 min. Membranes were then washed thoroughly and exposed to a peroxidase substrate (detection buffers C and D; RayBiotech, Inc., Norcross, GA) for 5 min in the dark before imaging. Membranes were exposed to X-ray film (Kodak X-Omat AR) within 30 min ofaddition of the substrate. Signal intensities were quantified with Bio-Rad VersaDoc Imaging System 3000 and analyzed with Quantity One software (Bio-Rad). Biotin-conjugated immunoglobulin G served as a positive control at six spots, where it was used to identify membrane orientation and to normalize the results from different membranes that were being compared. For each spot, the net optical density level was determined by subtracting the background optical level from the total raw optical density level.
Sample preparation for PowerBlot and Western blot analysis. Cells were grown in 10-cm-diameter tissue culture plates and treated with live P. gingivalis, P. gingivalis LPS, or FimA for 24 h. Whole-cell lysates were prepared as follows: after removal of the medium by aspiration, cells were rinsed with phosphate-buffered saline three times. Boiling lysis buffer (10 mM Tris [pH 7.4], 1.0 mM sodium orthovanadate, and 1.0% SDS) was added to the plates to denature cellular proteins rapidly. The cell lysates were collected into a 50-ml conical tube and heated briefly in boiling water. The lysates were then sonicated for 10 s to shear genomic DNA. The protein concentration was determined with the Bio-Rad protein assay kit, and the samples were frozen at –80°C and sent on dry ice to BD Biosciences/Pharmingen (San Diego, CA) for PowerBlot analysis.
PowerBlot analysis. The levels of proteins were determined by PowerBlot analysis by BD Biosciences/Pharmingen (San Diego, CA). Briefly, samples containing 500 μg of protein in 500 μl of sample buffer were loaded in one large well on top of a 7.5 to 15% gradient SDS-polyacrylamide slab gel. Electrophoresis was conducted overnight at a constant current. The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). After transfer, the membranes were incubated in blocking buffer containing 5% nonfat dried milk. The membrane was clamped with a grid that isolates 40 lanes (chambers) oriented from the top to the bottom of the membrane. Mixtures containing four to five mouse monoclonal antibodies that recognize proteins of distinct molecular weights and antigenicities were added to each lane and allowed to hybridize for 1 h. A total of 995 antibodies were applied to seven templates per sample for this analysis. The blot was removed from the manifold, washed, and hybridized with goat anti-mouse antibody conjugated with horseradish peroxidase. The blots were developed with a chemiluminescence system (SuperSignal West Pico; Pierce, Rockford, IL). Each of the samples was analyzed on triplicate blots.
After exposure to X-ray films, the developed films were scanned, and the "trace" of each band was processed for densitometric analysis. Briefly, the trace includes a measure of both band intensity and band area. Standard average is the average trace for all bands on a blot. The individual band intensity was expressed as a percentage of the corresponding trace for average standards and for standards designated "percent lane average" to normalize for different exposures and protein loadings between gels. The percent lane average for bands of samples treated by P. gingivalis and its components (percent lane average for treated bands) was expressed as a percentage of the percent lane average for the corresponding control bands. The "percentage of control" was determined to express increases or decreases in protein expression. Changes are expressed as fold increase or decrease between the control percent lane average and the treated percent lane average. A summary file was provided, listing all protein expression changes detected in order of confidence, 1 through 5, with 5 being the highest level of confidence. The confidence level was based on fold change, reproducibility, and signal intensity (a good quality signal easily passes a visual inspection; it is hard for a low signal to pass a visual inspection). Levels are defined as follows: level 5, changes of >2-fold in triplicate from good quality signals; level 4, changes of 1.5- to 1.99-fold in triplicate from good quality signals; level 3, changes of 1.25- to 1.49-fold in triplicate from good quality signals; level 2, changes of >2-fold in triplicate from low signals; level 1, changes of >2-fold in duplicate from good quailty signals. In this article, we present only changes from levels 2 and higher.
Western blot analysis. Cell lysates prepared as described above or with cold lysis buffer (25 mM HEPES [pH 7.7], 400 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 0.5% Triton X-100, 3 mM dithiothreitol, 20 mM -glycerophosphate, 1mM sodium orthovanadate, and 25 mM para-nitrophenylphosphate and protease inhibitor mixture [Roche]) were analyzed by conventional Western blotting. Protein (50 μg) was electrophoresed through an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad) by electroblotting. The membranes were incubated in blocking buffer (5% nonfat dried milk, 10 mM Tris [pH 7.5], 100 mM NaCl, and 0.1% Tween 20). Immunoblotting for protein expression was performed with mouse monoclonal antibodies (BD Biosciences/Transduction Laboratories) against eight proteins selected from the proteins identified by the PowerBlot analysis. We used horseradish peroxidase-conjugated goat anti-mouse as a secondary antibody. The blots were developed with an enhanced chemiluminescence system (Amersham). Western blotting was repeated at least two times.
Cytokine ELISA. The supernatants from live P. gingivalis-, LPS-, or FimA-stimulated human macrophages were analyzed for TNF-, IL-6, and IL-10 by ELISA (R&D Systems, Minneapolis, MN).
Statistics. Statistical analysis was performed by the two-tailed Student t test for comparison between two groups. A P value of <0.05 was considered statistically significant. The correlation between two groups was quantified by use of the linear correlation coefficient.
RESULTS
Cytokine expression in human monocytes exposed to live P. gingivalis. To assess the impact of P. gingivalis on macrophage phenotype and function, freshly elutriated human monocytes were cultured as adherent monocyte-derived macrophages and exposed to live P. gingivalis. The cell culture supernatants were subjected to cytokine antibody array analysis (Fig. 1). In these isolated primary macrophage cultures, live P. gingivalis induced MCP-1, MIP-1, angiogenin, FGF-4, IGFBP-2, and PARC as early as 1 h after exposure (Fig. 1C). Then, GM-CSF, GRO, IL-6, IL-10, TNF-, MIP-3, and TIMP-2 were detected at 4 h, while ENA-78, GRO-, and IL-1 were detected at 24 h (Fig. 1D). In addition, among the earliest-induced cytokines, FGF-4 and PARC levels decreased or were not detected at 4 h, while MCP-1, MIP-1, MIP-3, and angiogenin levels decreased or were not detected at 24 h (Fig. 1C). NAP-2 and MDC were constitutively expressed in adherent monocyte-derived macrophages, but they were completely inhibited or degraded by live P. gingivalis after 24 h (Fig. 1B and C).
Differential effects of P. gingivalis and its purified components on the induction of cytokines in human monocytes. Following the initial infection, P. gingivalis can release its cell surface components such as LPS and FimA, which contribute to the progression of periodontitis. To establish whether P. gingivalis stimulated a profile of cytokine expression different from its LPS, compared to its FimA protein, in human monocytes, cultured monocyte-derived macrophages were treated with intact microorganisms, purified LPS, or purified FimA for 24 h and cytokine induction detected by cytokine array was compared with that of parallel unstimulated cell populations (Fig. 2). In these macrophage cultures, untreated controls were found constitutively to express IL-8, PARC, MCP-1, MDC, MIP-1, angiogenin, NAP-2, and TIMP-2 (Fig. 2A), while all of the other cytokines in the array were undetectable. Expression levels of IL-6, IL-10, TNF-, MIP-3, MIP-1, and ENA-78 in macrophages exposed to live P. gingivalis, purified LPS, and FimA were similar at 24 h, with IL-6, IL-10, TNF-, and MIP-3 being the dominant cytokines, whether triggered by P. gingivalis or by its components. A constitutively expressed cytokine, PARC, was not detectable either with P. gingivalis or with its two purified components after 24 h of exposure. However, expression of IL-1, GM-CSF, GRO-, and IGFBP-2 was induced only by live P. gingivalis, whereas, inversely, expression of IP-10, RANTES, MCP-1, MCP-2, MCP-3, OSM, and TGF-2 was induced only by its purified components LPS and FimA (Fig. 2B and C). MCP-1 was induced by P. gingivalis after a 4-h exposure but was down-regulated after 24 h of exposure (Fig. 1). Both P. gingivalis and its FimA protein induced I-309/CCL-1, while LPS induced IGFBP-3. TIMP-2 was up-regulated by live P. gingivalis but down-regulated by the purified LPS.
In order to check the reliability of the relative differences observed between cultures treated with P. gingivalis and with its components, we quantified the levels of TNF-, IL-6, and IL-10, three cytokines that showed large fold differences in levels among cultures. The actual TNF-, IL-6, and IL-10 levels in the cell supernatants used in the antibody arrays were determined by a conventional ELISA, as described in Materials and Methods. The actual TNF-, IL-6, and IL-10 levels (in pg/ml) showed the same patterns seen in the arrays (Table 1). The levels of TNF-, IL-6, and IL-10 detected by ELISA correlated well with the net optical intensity differences seen in the antibody array experiments. The linear correlation coefficients (r values) for TNF-, IL-6, and IL-10 were 0.9911, 0.9936, and 0.9962, respectively.
Identification of differentially expressed proteins induced by P. gingivalis or its purified components as revealed by PowerBlot analysis. Extracts of human monocyte-derived macrophages grown for 24 h in control medium or in media supplemented with live P. gingivalis, LPS, or FimA were subjected to PowerBlot analysis. A representative blot of proteins extracted from untreated cells is shown in Fig. 3A. Enlarged parts of this control blot compared with corresponding regions from blots derived from P. gingivalis-treated or cell surface component-treated cells demonstrating changes (increases or decreases) in the levels of several proteins (circled or enclosed within rectangles) are presented in Fig. 3B through J. The analysis of the PowerBlot antibody array data indicated that P. gingivalis and its purified components induced both increases and decreases in the levels of specific proteins. Thus, treatment of the human macrophages with live P. gingivalis, its LPS, or its FimA protein led to up-regulation of 12, 8, and 10 proteins and down-regulation of 15, 8, and 17 proteins, respectively (Table 2). Included in this table are proteins involved in cell signaling, cell adhesion, cell growth, gene transcription, DNA repair, apoptosis, and other fundamental cellular processes. The changes induced in protein levels by P. gingivalis and its purified LPS and FimA could be categorized into several types: (i) changes that were similar for the three stimuli, (ii) changes that were induced by two of the three stimuli, (iii) changes that were induced by only one of the stimuli, and (iv) changes that were modulated uniquely by each stimulus.
Validation of PowerBlot results by conventional Western blotting. To confirm the PowerBlot data, we selected eight proteins (ILF3, STAT1, MAPK3, MEF2D, TNFSF6, CALR, STAT6, and PRKR) with various levels of differential expression and representing different confidence levels of changes detected by PowerBlot for analysis by conventional Western blotting (Fig. 4). With one exception (LPS-induced MAPK3), the conventional Western blotting analysis confirmed the differential expression of these eight proteins, which are listed in Table 3. The amounts of MEF2D, CALR, and PRKR induced by LPS relative to controls increased 2.03-, 2.54-, and 3.64-fold, respectively (Fig. 4), concordant with their 2.62-, 2.98-, and 3.7-fold differential expressions observed in the PowerBlot analysis results (Table 2). The amounts of ILF3, TNFSF6, CALR, PRKR, STAT1, and MAPK3 induced by FimA relative to controls increased 1.53-, 1.68-, 5.62-, 1.87-, 0.64-, and 0.37-fold, respectively (Fig. 4), in good agreement with their 1.43-, 2.22-, 2.98-, and 5.59-fold increases and 3.35- and 2.31-fold decreases, respectively, observed in the PowerBlot analysis results (Table 2). The relative ratios of STAT1, MAPK3, STAT6, PRKR, and CALR to controls induced by P. gingivalis were 0.08, 0.43, 0.32, 0.66, and 4.21, respectively (Fig. 4); these are comparable with the 16.63-, 4.20-, and 3.46-fold decrease and the 6.15-fold increase in differential expression observed for these proteins, respectively, in the PowerBlot analysis. MAPK3 induced by LPS was the only exception that was observed: its levels decreased according to the Western blot analysis but were unchanged according to the PowerBlot analysis. P. gingivalis, its purified LPS, and FimA all increased CALR expression in human monocytes in the PowerBlot analysis. This was also confirmed by two-dimensional gel electrophoresis followed by mass spectrometry (data not shown).
DISCUSSION
The innate immune response to P. gingivalis is initiated when the microorganisms interact with cell surface pattern receptors, including TLRs, to signal and/or become internalized. Recognition occurs largely through the bacterial cell surface constituents, such as LPS and fimbriae, and those cell surface constituents bind to TLR2 and also may bind to the glycosylphosphatidylinositol-linked protein CD14 (9, 10, 48) to induce inflammatory responses. It is thought that many of the effects of bacteria on host immune and inflammatory responses are mediated by changes in gene transcription. Indeed, changes in gene expression have been identified in cells treated with P. gingivalis and its purified LPS or fimbriae using differential display (35) and cDNA array (15) techniques, both of which are focused on mRNA analysis. Changes at the mRNA level may not correspond to changes in protein levels or to changes in activity, especially when those depend on posttranslational modifications and/or protein stability. Therefore, it is important to investigate the effects of P. gingivalis and its components on protein levels. Thus far, few reports have addressed the effects of P. gingivalis and its components on the modulation of proteins in human peripheral monocytes using large-scale proteomics. We designed this study to compare the effects of P. gingivalis and its components to see whether they cause distinct biological effects in human peripheral monocyte-derived macrophages. This article provides the first description of host proteins by P. gingivalis and its components LPS and FimA.
Using cytokine antibody arrays, we first investigated cytokines, the major inflammatory mediators induced by live P. gingivalis. At the initial interaction with macrophages, P. gingivalis induced chemokines (e.g., MCP-1, MIP-1, and PARC) involved in macrophage and neutrophil recruitment and in the antigen-specific T helper cell type 1 immune response (14, 47). However, this immune response may be suppressed after 24 h of exposure to P. gingivalis, since those chemokines, along with DMC/CCL22 and NAP-2, were dramatically reduced or disappeared completely after that time (Fig. 1 and 2). Following the initial response, expression of an extensive set of cytokines (including TNF-, IL-6, and IL-1) and other mediators transiently escalated subsequent to infection. Simultaneously, increases in IL-10 and TGF- may have contributed to the dampening of the initial activation response (3, 4). TNF- and IL-1, proinflammatory cytokines that activate multiple signal transduction pathways to influence both immune and nonimmune cell functions, are known to inhibit macrophage apoptosis (24). This agrees with previous observations that infection with P. gingivalis appears to protect macrophages from apoptosis (30, 33, 36, 50), thereby maintaining the pool of infected and infectible targets.
The immune and inflammatory responses to live bacteria may not be triggered simply by bacterial cell surface components, although these are among the best-characterized pattern recognition receptor ligands. Our cytokine antibody array data show that LPS and FimA induce similar patterns of cytokine production by human monocyte-derived macrophages; however, the patterns of cytokines produced by human macrophages stimulated by LPS or FimA differed substantially from that observed when the cells were challenged with live P. gingivalis (Fig. 2). It seems that the host defense system senses live bacteria differently than individual bacterial constituents, and thus it launches a different immune response. Our data are consistent with previous studies demonstrating that both P. gingivalis LPS and FimA signal through a common pattern recognition receptor, TLR2, whereas live P. gingivalis signals through both TLR2 and TLR4 in the induction of cytokine production by mouse peritoneal macrophages (9, 52).
In order to identify intracellular signaling pathways activated during P. gingivalis infection, we identified intracellular proteins modulated in human macrophages after interaction with live P. gingivalis and its by-products, LPS and FimA, by using the PowerBlot array screening system. The analysis demonstrates differences in the levels of several proteins after macrophage exposure to P. gingivalis, its LPS, or its FimA protein (Table 2). These proteins included those associated with immune response, both Ser/Thr and Tyr protein kinases, heat shock response proteins, transcription factors, DNA repair enzymes, and proteins engaged in vesicular transport, apoptosis, cell cycle, and cell adhesion. Additional studies are required to determine whether some or any of the changes in the levels of proteins are due to transcriptional regulation via ligand-specific receptors and whether they are the cause or consequence of the biological effects. It is noteworthy that we have analyzed changes that occurred after 24 h of cell treatment, and so some of the changes may be earlier than changes in cell cycle or cell viability. Here we have elected to highlight several of the modulated proteins that may be related to the mechanisms underpinning the effects of P. gingivalis and its constituents on infection, inflammation, and immune response.
We observed that similar changes in CLTC, CALR, RAB27A, and MAPK3 levels were induced by P. gingivalis, its LPS, and its FimA protein, suggesting a common mechanism of action. CLTC, a clathrin heavy-chain protein, was down-regulated after exposure to all three treatments, which may result in attenuated macrophage chemotaxis and phagocytosis (18, 46). A Ca2+ binding protein, CALR, which was reported to modulate the host immune response by binding to complement C1q, thereby participating in the inflammatory processes associated with vascular or atherosclerotic lesions, autoimmune diseases, or infections (7), was up-regulated by these three stimuli. The GTP-binding protein RAB27A, which appears to be a key effector of cytotoxic granule exocytosis, a pathway essential for immune homeostasis (28), was also down-regulated under all three conditions. P. gingivalis, its LPS, and its FimA protein all repressed the basal expression of MAPK3 (ERK1) in macrophages; suppression of MAPK3 was reported to facilitate an adaptive regulation in the process of cell proliferation under cell growth arrest conditions (11). All of these functions may favor its establishment and propagation within the host after P. gingivalis infection or enhance the host's chronic inflammatory reactions.
Purified LPS from P. gingivalis specifically induced MEF2D, IFIT4, and CDC42. MEF2D is a member of the myocyte enhancer factor 2 family of transcription factors and has been shown to increase CD14 expression in monocytes/macrophages (34); up-regulation of the MEF2D protein by P. gingivalis LPS may enhance macrophage responses to LPS by possibly increasing CD14 expression. That LPS up-regulates the interferon-inducible antiviral protein IFIT4 was also found by other investigators (16); however, its role in biological processes and molecular function here is still unknown and needs further investigation. The GTPase CDC42 is a GTP-binding protein that is involved in macrophage phagocytosis via the calcium-promoted Ras inactivator (51). However, P. gingivalis LPS may not induce phagocytosis and oxidative burst by activating CDC42, because we did not detect the up-regulation of the calcium-promoted Ras inactivator in macrophages treated with P. gingivalis LPS.
FimA has been shown to up-regulate TNFSF6 (CD95L) and ILF3. A type II transmembrane protein of the TNF family of death factors, TNFSF6 has shown to be associated with the establishment of immune privilege and tumor survival (39). Interaction between TNFSF6 and its receptor can regulate key events in cellular activation, proliferation, differentiation, cell death, and survival of immune cells and other tissues (22). ILF3 is a transcription factor required for T-cell expression of IL-2 (25). ILF3 binds to a sequence in the IL-2 enhancer known as antigen receptor response element 2. In addition, ILF3 can bind RNA and is an essential component for encapsidation and protein priming of hepatitis B viral polymerase (17). That FimA induced TNFSF6 and ILF3 expression may indicate that it plays an important role in affecting the host immune system.
Live P. gingivalis dramatically down-regulated expression of the transcription factor STAT6. This is an intriguing finding, in light of other data that STAT6 deficiency confers enhanced immunity against tumors (44) as well as against parasite infection (40, 43). STAT6-deficient mice generate excess nitric oxide in their macrophages compared to the macrophages from wild-type mice, which produce arginase instead of nitric oxide (44). Nitric oxide-producing macrophages have a strong ability to induce antigen-specific CD4+ T-cell proliferation in response to unrelated antigens (41). It seems that during the acute infection stage (24 h after infection), P. gingivalis turns on nitric oxide production by macrophages as a host defense mechanism, accomplished by down-regulating the expression of STAT6.
The level of STAT1 was decreased in macrophages treated with either FimA or live P. gingivalis. STAT1 plays an essential role in the caspase-independent cell death of activated macrophages through the p38 MAPK/STAT1/reactive oxygen species pathway (20). Decreased STAT1 levels were reported to decrease superoxide production in macrophages (20). Down-regulation of STAT1 by P. gingivalis or by its FimA protein may be one of the several survival mechanisms used by P. gingivalis to evade host defenses.
STAT3 is a transcription factor mediating the anti-inflammatory properties of IL-10 (49). It has been reported that mice with a STAT3 deficiency display excessive local and systemic inflammation and fail to facilitate bacterial clearance (27). We noticed that P. gingivalis LPS down-regulated and FimA up-regulated STAT3 expression in macrophages; this may indicates that LPS plays a negative role while FimA plays a positive role in stimulation of anti-inflammatory activity and bacterial clearance during P. gingivalis infection. In addition, the STAT3 level in macrophages was not changed by whole bacterial treatment compared to that of untreated macrophages; it seems that the whole bacteria balanced the role of LPS and FimA on inducing STAT3 expression in macrophages.
PRKR, an interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (p68 kinase), acts as a tumor suppressor by actively participating in apoptosis (21). PRKR is also essential for antiviral activity by inhibiting both viral and cellular protein synthesis (19). Interestingly, it was increased only by LPS and decreased by both live P. gingivalis and FimA. The abilities of live P. gingivalis and FimA to decrease PRKR levels may be important for the antiapoptotic activities observed by other investigators (50). However, the ability of P. gingivalis LPS to increase PRKR levels may not related to apoptogenic activity, because P. gingivalis LPS also possesses antiapoptotic properties (30).
Another interesting observation of this study was the down-regulation of TPL-2 (MAP3K8) by live P. gingivalis and its LPS. P. gingivalis LPS is a TLR2 agonist; it was reported to inhibit a TLR4 agonist, E. coli LPS-induced TNF- production, with IB kinase activity being unperturbed (6). This TLR "heterotolerance" induced by P. gingivalis LPS may come about through down-regulation of TPL-2, via an IB kinase-independent pathway, because TPL-2 is essential for TLR4 activation of the ERK MAPK cascade in E. coli LPS-stimulated macrophages and subsequent up-regulation of genes involved in innate immune responses (2). The down-regulation of TPL-2 may also be associated with the down-regulation of MAPK3 detected in this study, which could weaken the innate host immune responses to bacterial infection.
In summary, the large-scale screening of proteins modulated after treatment of human monocyte-derived macrophages with live P. gingivalis, its LPS, and its FimA protein has enabled us to identify proteins that are implicated in the effects of P. gingivalis and its components on the innate immune responses of human monocytes and macrophages. P. gingivalis and its components appear to stimulate different protein expression patterns in macrophages that may reflect intrinsic functional differences in these stimuli. This differential modulation of proteins in host cells may well coordinate with their specific roles in triggering host inflammatory/immune responses during P. gingivalis infection.
ACKNOWLEDGMENTS
This work was supported by grant RO1 DE 15989 from NIDCR.
Present address: The Pulmonary Center, School of Medicine, Boston University Medical Center, 15 Stoughton Street R-304, Boston, MA 02118.
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