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Identification of Signaling Pathways in Macrophage Exposed to Porphyromonas gingivalis or to Its Purified Cell Wall Components¹

Department of Periodontology and Oral Biology, School of Dental Medicine, Boston University, Boston, MA 02118

	Abstract

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Porphyromonas gingivalis (P. gingivalis) can trigger an inflammatory condition leading to the destruction of periodontal tissues. However P. gingivalis LPS and its fimbriae (FimA) play different roles compared with the live bacteria in the context of intracellular molecule induction and cytokine secretion. To elucidate whether this difference results from different signaling pathways in host immune response to P. gingivalis, its LPS, or its FimA, we examined gene expression profile of human macrophages exposed to P. gingivalis, its LPS, or its FimA. A comparison of gene expression resulted in the identification of three distinct groups of expressed genes. Furthermore, computer-assisted promoter analysis of a subset of each group of differentially regulated genes revealed four putative transcriptional regulation models that associate with transcription factors NFB, IRF7, and KLF4. Using gene knockout mice and siRNA to silence mouse genes, we showed that both TLR2 and TLR7 are essential for the induction of NFB-containing genes and NFB-IFN-sensitive response element (ISRE) cocontaining genes by either P. gingivalis or its purified components. The gene induction via either TLR2 or TLR7 is dependent on both MyD88 and p38 MAPK. However, the unique induction of IFN-β by P. gingivalis LPS requires TLR7 and IFNβR cosignaling, and the induction of ISRE-containing gene is dependent on the activation of IFN-β autocrine loop. Taken together, these data demonstrate that P. gingivalis and its components induce NFB-containing genes through either TLR2- or TLR7-MyD88-p38 MAPK pathway, while P. gingivalis LPS uniquely induces ISRE-containing genes, which requires IFNβR signaling involving IRF7, KLF4, and pY701 STAT1.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Porphyromonas gingivalis (P. gingivalis) is a predominant periodontal pathogen that colonizes periodontal pockets and then spreads into deeper tissues. Inflammation following P. gingivalis infection leads to the destruction of periodontal tissues, resorption of alveolar bone, and exfoliation of teeth (1). Various cellular components of P. gingivalis are thought to function as virulence factors, including LPS and fimbriae. LPS from P. gingivalis induces multiple biological and immunological activities through TLRs (2). It has also been shown to antagonize Escherichia coli LPS-dependent induction of E-selectin expression, p38 MAPK activation, and NF-B activation in human endothelial cells and monocytes (2, 3). The ability of P. gingivalis LPS to down-regulate innate immune responses to other LPS (E. coli) may play a role in allowing this oral pathogen to evade the normal surveillance system so essential in maintaining periodontal health (4). Fimbriae are reported to mediate the bacterial adherence to and invasion of epithelial cells and gingival fibroblasts (5). Although the LPS of P. gingivalis is reported to play the most critical role in inducing proinflammatory responses in infected periodontal tissues, several investigators have suggested that fimbriae of this pathogen can trigger the production of proinflammatory mediators in human endothelial and epithelial cells and macrophages (5, 6, 7). Therefore, these bacterial factors are likely to contribute differentially in the progression of the overall inflammatory destruction of infected periodontal tissues.

The innate immune system is the body’s first line of defense against infection. The outcome of infection is the net consequence of the immune defenses of the host and a pathogen’s capacity to subvert them. Macrophages play a central role in regulating innate and acquired immune responses against pathogens: they identify foreign invaders using pattern-recognition receptors, such as TLRs, which detect highly conserved microbial-specific structures. Macrophages, once activated via TLRs, unfold a tightly controlled pathogen-specific immune response (8). The mechanisms by which macrophages interact with P. gingivalis are complex and involve the coregulation of specific signaling and transcriptional machinery in response to this bacterium and to its cell surface components (2, 5). Achieving a better understanding of the molecular basis of host response to P. gingivalis will be critical for preventing periodontal infection and also for minimizing the tissue damage resulting from an overly aggressive host response.

Our previous study demonstrated qualitative and quantitative differences in the response of macrophages to P. gingivalis compared with its fimbriae or LPS (7, 9, 10), supporting our hypothesis that unique signaling mechanisms are induced by P. gingivalis vs its components, and these differential signaling may play important roles in P. gingivalis acute vs chronic infection. Indeed, in acute infection, the host is sensing mostly live bacteria, whereas in chronic infection, a combination of live bacteria and subsequent breakdown of its cell wall (i.e., LPS, fimbriae) by host immune cells. To improve our understanding of the mechanisms by which macrophages interact with P. gingivalis, we attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).³ We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages. Our study demonstrates that there is an overlapping pattern of regulated genes in human macrophages representing a general inflammatory response to P. gingivalis and to its cell surface components. Additionally, P. gingivalis and its purified components preferentially induce regulation of private sets of genes that may specifically perturb the macrophage response to infection. Pathway analysis further demonstrates that P. gingivalis and its components commonly activate NFB-containing genes through either TLR2- or TLR7-MyD88-p38 MAPK pathway, while purified P. gingivalis LPS uniquely induces IFN-sensitive response element (ISRE)-containing genes requiring IFNβR signaling.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Bacterial strain and reagents

P. gingivalis 381 (American Type Culture Collection) was cultured anaerobically, as described previously (9). Protein-free LPS from P. gingivalis 381 was extracted with phenol-water and purified by cesium chloride isopyknic density gradient ultracentrifugation followed by repurification, and FimA was purified by size exclusion chromatography, as previously described (9). Abs against IRF3, IRF7, KLF4, p65 NFB, STAT1 (including pY701 and pS727), and β-actin, along with HRP-conjugated anti-rabbit or anti-goat Ig G, were obtained from Santa Cruz Biotechnology. Ficoll-Hypaque (1.0771) was purchased from Sigma-Aldrich; Monocyte Isolation Kit II, FITC-conjugated anti-CD14, and PE-conjugated anti-biotin were purchased from Miltenyi Biotec; RPMI 1640, DMEM, FBS, Gentamicin, penicillin/streptomycin, and Lipofectamine 2000 reagents were purchased from Invitrogen Life Technologies; human serum type AB was purchased from Cambrex; and RNeasy Mini Kit was obtained from Qiagen.

Animals

C57BL/6 mice were purchased from The Jackson Laboratories. TLR2 knockout (TLR2^–/–), MyD88 knockout (MyD88^–/–), and TLR7 knockout (TLR7^–/–) mice on a C57BL/6 background were a gift from Dr. S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan). All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Boston University Medical Center.

Macrophage cultures

Human PBMC were derived by Ficoll-Hypaque density gradient centrifugation from buffy coats of different healthy blood donors obtained from the Interstate Blood Bank (Memphis, TN). All blood donors are male, 25 to 45 years old, and were evaluated by FDA-licensed testing procedures to eliminate donors whose specimens indicated the presence of HIV, hepatitis B virus, hepatitis C virus, or syphilis infections. CD14-positive monocytes were enriched from PBMC to >95% purity by immunomagnetic elimination of T cells, NK cells, B cells, dendritic cells, and basophils using the Monocyte Isolation Kit II. Purity of monocytes was determined by flow-cytometric analysis of FITC-conjugated anti-CD14 and PE-conjugated anti-biotin. Purified human monocytes were plated at a density of 2 x 10⁶ cells/ml in DMEM with 20% FBS, 10% human serum AB, and 50 µg/ml gentamicin in 6-well or 10-cm diameter tissue culture plates for 5 days at 37°C, in a humidified atmosphere containing 5% CO₂. On days 5 and 7, half of the medium were removed and replaced with medium lacking FBS. Media on the cultured macrophages were replaced with fresh DMEM containing 1% human serum on day 9, 1 hour before experiments were begun.

Mouse peritoneal macrophages were isolated by peritoneal lavage, as described previously (9). Isolated macrophages were plated into 6-well plates at a concentration of 1 x 10⁶ cells/ml in RPMI 1640 medium supplemented with 10% FBS and standard penicillin/streptomycin. After a 2 h-incubation at 37°C in an atmosphere containing 5% CO₂, nonadherent cells were washed out with warm PBS. Adherent macrophages were cultured for 2 days before experiments. Media was changed 1 h before experiments began.

Infection of macrophages with P. gingivalis and treatment of macrophages with P. gingivalis components

Adherent macrophages were infected with live P. gingivalis at indicated multiplicities of infection (MOI). Live P. gingivalis 381 frozen stocks were thawed and cultured for 24 h, and then the cultures were collected and diluted in medium to a concentration of 5 x 10⁸ bacteria per 50 µl, to give MOIs as indicated, and added to cultures of macrophages. Dilutions were also plated on brain-heart infusion agar plates for anaerobic culture, and colonies were counted to confirm the accuracy of dilution and viability of bacteria. In additional cultures, purified LPS or FimA from P. gingivalis were added to cell culture medium at indicated concentrations. Cells were incubated at 37°C in an atmosphere containing 5% CO₂. Cells and supernatants were harvested at indicated times after incubation with live P. gingivalis or its components.

RNA preparation and Affymetrix GeneChip microarray analysis

Two hours after infection with P. gingivalis, or treatment with P. gingivalis LPS, FimA, or saline (control), human macrophages were washed three times with ice cold PBS, and total RNA was extracted using an RNeasy Mini Kit according to the manufacturer’s instructions. For each condition, three independent experiments were performed. For each experimental sample, RNA quality was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Concentrations were also determined using a NanoDrop 1000 spectrophotometer. Under standard conditions, processing of RNAs for GeneChip Analysis was conducted in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision 4, as subsequently detailed. Synthesis of cDNA first and second strand were performed using the GeneChip Expression 3'-Amplification Reagents One-Cycle cDNA Synthesis Kit (P/N 900431). In vitro transcription was performed using the GeneChip Expression Amplification Reagents kit-30 reactions (P/N 900449), and was conducted according to the standard Affymetrix protocols. Hybridization was conducted according to the Affymetrix GeneChip Manual. Twenty micrograms of in vitro transcription material were used on each GeneChip Human Genome U133 Plus 2.0 array, which contains 54674 gene probe sets. Affymetrix hybridization ovens were used to incubate the arrays overnight at a constant temperature of 45°C. Preparation of microarrays for scanning was conducted with Affymetrix wash protocols appropriately matched to the specific chip type on a Model 450 Fluidics station. Affymetrix GeneChip Operating Software operating system controls the Fluidics station process. Scanning was conducted on a GeneChip Scanner 3000 7G scanner with autoloader. The Affymetrix GeneChip Operating Software v1.3 operating system controlled the Model 3000 7G scanner and data acquisition functions and maintained the mediated first-level data analysis and desktop data management for the entire GeneChip System.

Data processing

Primary data from the microarray experiments were analyzed algorithmically using ArrayAssist software (Version 4.1.0; Stratagene). Fluorescence intensities were first normalized to median array intensities for all conditions tested, and then each intensity value was converted to its log base 2 value. The fold changes were calculated relative to unstimulated baseline controls. Data from three independent replicate experiments were used to perform a paired two-sample t test for each gene. Data from a total of 12 arrays (three treated conditions and one untreated condition with three replicates for each condition) were included in the analysis. A filter for regulated genes used the following stringent criteria to define genes as significantly differentially expressed: 1) fold change of 2 or –2, which signifies changes in the expression level between control conditions (baseline) and stimulated cells; 2) a change in the p value of <0.05, which describes the likelihood of change of expression for each transcript, where p values indicated the level of significance of the difference between the baseline and experimental conditions based on the paired two-sample t test; and 3) absolute difference in signal intensity between group means of 50. Hierarchical clustering algorithms were used to group together genes with similar expression patterns. Pathway Architect Software (Stratagene) was used for gene ontology assessment and pathway visualization.

Computer-assisted promoter analysis

Promoter sequences 1000 bases upstream from the transcriptional start site of selected genes were obtained from Advanced Biomedical Computing Center (ABCC) (http://grid.abcc.ncifcrf.gov/promoters.php). Gene promoters were assessed for potential transcription factor (TF) binding sites using the Transcription Regulatory Element Search program (http://bioportal.bic.nus.edu.sg/tres). The input sequences can be searched for conserved TF binding sites using nucleotide frequency distribution matrices described in the TRANSFAC database (11). The position weights and matrix similarity scores are essentially calculated according to Quandt et al. (12). A TF binding site is considered conserved only when the matrix similarity score is 90. The output matrix positions correspond to sense strand numbering, and all sequences are provided in the 5'-3' direction. The National Center for Biotechnology Information gene accession numbers of selected genes that were analyzed are as follows: TSLP (NM_033035), CXCL3 (NM_002090), PTGS2 (NM_000963), PTX3 (NM_002852), IL12B (NM_002187), CXCL1 (NM_001511), CXCL10 (NM_001565), CCL20 (NM_004591), IL1B (NM_000576), TNF (NM_000594), IFN-β (NM_002176), SOCS1 (NM_003745), IRF7 (NM_004031), CXCL11 (NM_005409), IFIT1 (NM_001001887), IFIT2 (NM_001547), OASL (NM_003733), USP18 (NM_017414), ACSL4 (NM_022977), PBEF1 (NM_005746), and MAML2 (NM_032427). These genes were selected as representative of three groups of genes that are either commonly induced by P. gingivalis and by its cell surface components or uniquely induced by LPS and by P. gingivalis.

Quantitative real-time PCR (qRT-PCR)

Reverse transcription (RT) of total RNA (1 µg) in each 20 µl reaction was conducted using iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer’s instructions. RT reactions without iScript Reverse Transcriptase were used as negative controls. The reaction mixtures were diluted to 200 µl (10x dilutions) after RT, and 5.0 µl of each cDNA mixture was used for quantitative PCR. Primers for target genes were generated using Beacon Designer software (Bio-Rad). Human gene primers were as listed in Table I, and mouse gene primers were as follows: TLR2 (sense 5-ACTGTCCTGTGATACTGTTCTG, antisense 5-TGTGCCTGGTCTGTGTCC); TLR7 (sense 5-AGCCCTTTACCTGGATGGAAAC, antisense 5-CGTGATGGAGAAGATGTTGTTAGC); MyD88 (sense 5-AGCAGCAGAACCAGGAGTC, antisense 5-GGGCAGTAGCAGATAAAGGC); IFNβR (sense 5-AGGTGTTGTGTTCTTCTCTGTC, antisense 5-CCGTGTCTGTATTCTCAATGATG); PTX3 (sense 5'-AAGAATGGTTGCTGTGTAGGTG, antisense 5-CGCCTGAATCTCTGTGACTCC); CXCL10 (sense 5-TTCTGCCTCATCCTGCTG, antisense 5-AGACATCTCTGCTCATCATTC); IFN-β (sense 5'-GCTTCCTGCTGTGCTTCTC, antisense 5-CATCTTCTCCGTCATCTCCATAG); IFIT2 (sense 5'-GCCATTCAACTGTCTCCTG, antisense 5-GCTCTGTCTGTGTCATATACC); and β-actin (sense 5'-TTGACCAGAGCAGGCAGATG, antisense 5'-CTACCAGAAGGGCAGGATACAG). Quantitative PCR was conducted using the iQSYBR Green Supermix (Bio-Rad) in a Bio-Rad iCycler according to the manufacturer’s protocol. The mRNA expression of all samples was normalized to that of β-actin. The cycle threshold (C_T) value indicates the number of PCR cycles that are necessary for the detection of a fluorescence signal exceeding a fixed threshold. The fold change (FC) was calculated by using the following formulas: C_T = C_T (β-actin)–C_T (target gene) and FC = 2^(C_T2^–C_T1⁾, in which C_T1 represents the mean for P. gingivalis-, FimA- or P. gingivalis LPS-treated cells, and C_T2 represents the mean for control cells.

For analysis of MAPK phosphorylation, human macrophages were stimulated with live P. gingivalis, its LPS, or its FimA for 30 min and then solubilized at 1 x 10⁷ cells/ml in Lysis Buffer 6 (R&D Systems), according to the manufacturer’s instructions. Cell lysates were centrifuged at 14,000 x g for 5 min, supernatants were transferred into clean tubes, and protein concentrations were determined using a Bio-Rad Bradford protein assay. Phosphorylation of 21 MAPK was assayed simultaneously using Human Phospho-MAPK Array Kit (R&D Systems), according to the manufacturer’s instructions.

siRNA transfection

siRNAs for IFNβR, IFN-β, and TLR7 were synthesized by Ambion (Austin, TX) and targeted exons 6 and 7 of the human IFNβR gene (NM_000874, NM_207584, and NM_207585), exon 2 of the mouse IFNβR gene (NM_010508), exon 1 of the mouse IFN-β gene (NM_010510), and exon 3 of the mouse TLR7 gene (NM-133211), respectively. Ambion’s Silencer Negative Control siRNA was used to demonstrate that the transfection does not induce nonspecific effects on gene expression. For transient transfections of siRNA, each pair of oligoribonucleotides was annealed at a concentration of 40 µM and introduced into macrophages derived from human peripheral monocytes or mouse peritoneal macrophages in 6-well plates by transfection with Lipofectamine 2000 reagents, according to the manufacturer’s protocol. Forty-eight hours after transfection, cells were treated with P. gingivalis, its LPS, or its FimA for the indicated time, and total RNA, or protein, was extracted as described above.

Western analysis

Cell lysates were prepared 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 DTT, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 25 mM para-nitrophenylphosphate and protease inhibitor mixture; Roche). Protein concentrations were determined using a Bio-Rad Bradford protein assay. Proteins (50 µg) were 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) before immunoblotting was performed with primary Abs. HRP-conjugated Abs were used as secondary Ab. The blots were developed with an ECL system (Amersham Biosciences). Western blotting was repeated three times.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Differential gene induction in primary macrophages exposed to P. gingivalis, its LPS, or its FimA

Our previous studies have demonstrated that the response of macrophages to live P. gingivalis differs from their response to this bacterium’s cell surface components, LPS and FimA (9, 10). To further uncover the cellular signaling events responsible for these differences, we conducted a whole genome-based transcriptional analysis. This analysis examined the early response of human macrophages to P. gingivalis infection and to its LPS or its FimA treatment. First, we purified human monocytes from buffy coats of healthy blood donors, and the purity of monocytes was found to be over 95%, as determined by FACS analysis (Fig. 1, A and B). Because TNF- release is an important response of monocytes/macrophages to infections and because the extent of such release is correlated to bacterial virulence, we then monitored the TNF- levels to determine the optimal quantities of P. gingivalis, its LPS and its FimA for the in vitro experiments. The MOI of 25:1 was selected for P. gingivalis infection in vitro, because this ratio is equivalent to the potency of 10 µg/ml of either LPS or FimA, which is sufficient to induce TNF- in macrophages (Fig. 1, C and D).

View larger version (45K): [in this window] [in a new window]   FIGURE 1. TNF- levels in human monocyte-derived macrophages in response to P. gingivalis, its LPS, or its FimA. A, Peripheral blood PBMC were isolated by Ficoll-Hypaque density gradient centrifugation and subjected to FACS analysis using anti-biotin Ab and anti-CD14 Ab. B, Monocytes were purified from PBMC and subjected to FACS analysis using anti-Biotin Ab and anti-CD-14 Ab. C, Dose-dependent TNF- levels of macrophages were measured by ELISA in response to LPS and FimA. D, Dose-dependent TNF- levels of macrophages were measured by ELISA in response to live P. gingivalis.

Gene expression is regulated by nuclear transcription factors. The identification of three groups of genes among P. gingivalis and its components-simulated macrophages indicates that different transcriptional regulation patterns may exist in macrophages in response to P. gingivalis infection and to its purified components treatment. To determine whether unique transcriptional regulation patterns exist in different groups of genes, sequences of 1000 bases upstream from the transcriptional start site of selected genes from each of the groups were analyzed using the Transcription Regulatory Element Search program to identify common TF sites for each group. Four different TF site patterns were identified from the three groups of genes (Fig. 2A). All genes in Group 1 possess the NFB binding site in their promoters, but have no ISRE element (except CXCL10); therefore, they are defined as NFB-containing genes. Among these NFB-containing genes, some possess an NFB, but no Kruppel-like factor (KLF) site (Pattern I), while others possess both NFB and KLF binding sites (Pattern II) in their promoters. All genes in Group 2 have the ISRE element. Among these, some possess NFB site with or without KLF sites (Pattern III), which are defined as NFB and ISRE cocontaining genes; others possess KLF site, but no NFB site (Pattern IV), and these are defined as ISRE-containing genes. Genes in Group 3, such as ACSL4, PBEF1, and MAML2, have no common TF pattern and are unclassified (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 2. NFB, KLF4, and IRF7 were identified as potential transcription factors that regulate gene expression in response to P. gingivalis and its components. A, Promoter analysis reveals ISRE elements and NFB or KLF binding site in the upstream no-coding regions of selected genes differentially expressed in macrophages treated by P. gingivalis and its components. Elements 1000 bases 5' upstream from the transcriptional start site were analyzed by Transcription Regulatory Element Search to identify the presence of transcription factor binding sites. The nucleotide sequences and specific locations of DNA binding elements within these promoter regions are provided with positions relative to the transcriptional start site, which is defined as + 1. Symbols indicating distinct DNA binding elements (e.g., ISRE, NFB, KLF, STAT, AP-1, GC box, and TATA box) are shown. B, Depicted here as a heat map are transcription factors that were identified as being regulated by P. gingivalis, its LPS, and its FimA. Color intensity correlates with average log base 2 values of fluorescence intensities (n = 3; red corresponds to up-regulation and green corresponds to down-regulation). Transcription factors were grouped and then extracted with hierarchical clustering algorithms. Numbers under fold change are mean values of three independent experiments: –, down-regulated; NC, no change.

RELA (p65 NFB)

NFB

Role of TLR2 and TLR7 in macrophage gene induction by P. gingivalis, its LPS, or its FimA

Previous studies have demonstrated that P. gingivalis, its LPS, and its FimA activate macrophages via TLR2 (9, 13) or TLR4 (14). Macrophages then use TLRs to initiate a tightly controlled pathogen-specific immune response (15). Our cDNA microarray data showed that TLR2 and TLR7 were up-regulated by P. gingivalis, its LPS, or its FimA, while TLR1, TLR6, and TLR10, which are thought to associate with TLR2 to form heterodimers (16, 17, 18, 19), were down-regulated (Fig. 3A). To clarify the roles of TLR2 and TLR7 in gene induction by P. gingivalis, its LPS, and its FimA, we further used qRT-PCR to analyze gene expression for PTX3 (NFB-containing), CXCL10, IFN-β (NFB/ISRE con-containing), and IFIT2 (ISRE-containing) in TLR2^–/– macrophages as well as in TLR7 siRNA knockdown macrophages. The gene expression of TLR2 and TLR7 was confirmed by RT-PCR using total RNA from TLR2^–/– macrophages and TLR7 siRNA knockdown macrophages (Fig. 3B). The results of qRT-PCR, which corresponded to the cDNA microarray results, demonstrated that PTX3 and CXCL10 were induced by P. gingivalis, its LPS, or its FimA, while IFN-β and IFIT2 were induced by LPS alone (Fig. 3C). TLR2^–/– or TLR7 knockdown caused remarkable suppression of PTX3 and CXCL10 mRNA induction by all three stimuli, but little effect of TLR2 or TLR7 on the induction of IFN-β and IFIT2 by LPS was observed. Codeficiency of TLR2 and TLR7 severely impaired PTX3, CXCL10, and IFN-β mRNA induction, but exhibited a significantly lesser effect on IFIT2 mRNA induction (Fig. 3C). Because the activation of TLR7 by P. gingivalis, its LPS, or its FimA was an intriguing finding, we further used TLR7^–/– mice to confirm these results. As anticipated, TLR7 mRNA in TLR7^–/– macrophages was undetectable, but it was easily detected in wild type macrophages (data not shown). The induction of PTX3 and CXCL10 mRNA by live P. gingivalis, its LPS, or its FimA was dramatically attenuated in TLR7^–/– macrophages as compared with their wild type counterpart (Fig. 4, A and B), similar to the results as observed with TLR7 knockdown. In addition, the induction of IFN-β mRNA by LPS was also significantly reduced in TLR7^–/– macrophages (Fig. 4C), but the mRNA expression of IFIT2 was unimpaired (Fig. 4D). Together, these results indicate that activation of either TLR2 or TLR7 by P. gingivalis, its LPS, or its FimA are essential for the induction of both NFB-containing gene (PTX3) and NFB-ISRE cocontaining genes (IFN-β and CXCL10), but the induction of ISRE- containing gene (IFIT2) is largely independent of either TLR2 or TLR7.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Roles of TLR2 and TLR7 in macrophage gene induction by P. gingivalis, its LPS, and its FimA. A, A heat map shows a group of cell surface receptors that were regulated in macrophages by P. gingivalis, its LPS, and its FimA. Color intensity and map keys are described as in Fig. 2B. B, An agarose gel electrophoresis shows an RT-PCR analysis of TLR2 and TLR7 mRNA expression in wild-type (WT), TLR2–/–, and TLR7 siRNA-treated macrophages. C, WT, TLR2–/–, and TLR7 siRNA-treated macrophages were exposed to P. gingivalis, its LPS, or its FimA for 2 h, and the mRNA expression of PTX3, CXCL10, IFNB1, and IFIT2 in macrophages was measured by qRT-PCR using total RNA. The data shown are the average fold changes of three independent experiments. Asterisks indicate statistically significant (p < 0.05) differences in change of mRNA expression compared with wild-type control.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Gene induction by P. gingivalis, its LPS, or its FimA in TLR7–/– macrophages. Peritoneal macrophages from TLT7–/– and WT mice were exposed to P. gingivalis, its LPS, or its FimA for 2 h, and the mRNA expression of PTX3, CXCL10, IFNB1, and IFIT2 was measured by qRT-PCR using total RNA. The data shown is the average fold changes of three independent experiments. Asterisks indicate statistically significant (p < 0.05) differences in change of mRNA expression compared with WT control.

Role of IFNβR and MyD88 in macrophage gene induction by P. gingivalis, its LPS or its FimA

IFNβR has been demonstrated to play an important role in IFN-inducible gene induction (20). In addition, MyD88 is an adopter activated by both TLR2 and TLR7 and is essential for the induction of both inflammatory cytokine (21, 22) and type-I IFN production (23). Therefore, we compared the role of IFNβR and MyD88 in the induction of cytokine genes and IFN-inducible genes. The gene expression of IFNβR in siRNA knockdown macrophages and MyD88 expression in MyD88^–/– macrophages were either significantly suppressed or completely disappeared, respectively (Fig. 5A). Knockdown of IFNβR by siRNA had no effect on the suppression of PTX3 expression, but had remarkable effect on the suppression of CXCL10, IFN-β, and IFIT2 expression (Fig. 5B). However, stimulation of MyD88^–/– macrophages with P. gingivalis, its LPS, or its FimA was unable to activate PTX3 and CXCL10 gene expression, and the induction of IFN-β, but not IFIT2 by LPS, was severely impaired in MyD88^–/– macrophages (Fig. 5B). Codeficiency of IFNβR and MyD88 resulted in complete suppression of PTX3, CXCL10, IFN-β, and IFIT2 induction (Fig. 5B). Thus, these data indicate that the induction of PTX3 is dependent on MyD88, but not on IFNβR; the robust induction of CXCL10 and IFN-β is dependent on both MyD88 and IFNβR, while the induction of IFIT2 is dependent on IFNβR. To determine whether IFNβR plays the same role in human macrophages as in mouse macrophages, we analyzed the effect of IFNβR gene knockdown by siRNA on PTX3, CXCL10, IFN-β, IRF7, and IFIT2 gene induction in human macrophages. Our results showed that gene knockdown of IFNβR dramatically suppressed CXCL10, IFN-β, IRF7, and IFIT2 gene induction, but not PTX3 gene induction by LPS (Fig. 5C), confirming the role of IFNβR on IFN-inducible gene expression in human macrophages.

View larger version (46K): [in this window] [in a new window]   FIGURE 5. Roles of IFN/βR and MyD88 in macrophage gene induction by P. gingivalis, its LPS, or its FimA. A, An agarose gel electrophoresis shows an RT-PCR analysis of MyD88 and IFN/βR mRNA expression in wild-type (WT), MyD88–/–, and IFN/βR siRNA-treated macrophages. B, WT, MyD88–/–, and IFN/βR siRNA-treated macrophages were exposed to P. gingivalis, its LPS, or its FimA for 2 h, and the mRNA expression of PTX3, CXCL10, IFNB1, and IFIT2 was measured by qRT-PCR using total RNA. C, Human macrophages transfected with IFN/βR siRNA were stimulated with P. gingivalis LPS for 2 or 4 h, and the mRNA expression of PTX3, CXCL10, IFNB1, IRF7, and IFIT2 was measured by qRT-PCR using total RNA. The data shown is the average fold changes of three independent experiments. Asterisks indicate statistically significant (p < 0.05) differences in change of mRNA expression compared with control siRNA.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. LPS-induced IFN-inducible gene expression requires IFN-β. Peritoneal macrophages from C57BL/6 mice were transfected with siRNA for IFN-β gene and stimulated with P. gingivalis LPS for 4 h. Gene expression of PTX3 (A), IFN-β (B), CXCL10 (C), and IFIT2 (D) was analyzed by qRT-PCR using total RNA isolated from macrophages. The data shown is the average fold changes of three independent experiments. Asterisks indicate statistically significant (p < 0.05) differences in change of mRNA expression.

MyD88 is essential for p65 NFB phosphorylation, while IFNβR is required for the induction of KLF4 and IRF7 in macrophage response to P. gingivalis, its LPS, or its FimA

Transcription factors, such as NFB, KLF4, IRF3, and IRF7, have been reported to play an important role in regulating cytokine and IFN-inducible gene expression in mammalian cells (23, 24, 25). Based on our cDNA microarray and the computer-assisted promoter analysis of TF sites, the data presented clearly suggest that the transcription factors NFB, KLF4, and IRF7 do play an important role in gene induction by P. gingivalis, its LPS, or its FimA. Thus, we first assessed the role of TLR2 and TLR7 in the regulation of NFB p65, KLF4, IRF3, and IRF7 at the protein level. Neither P. gingivalis nor its components induced total p65 expression (data not shown), but all of them induced the phosphorylation of p65 NFB. Furthermore, the phosphorylation of p65 NFB was significantly suppressed by TLR2 knockout, but not by TLR7 siRNA (Fig. 7A). Expression of KLF4 and IRF7 was only induced by P. gingivalis LPS, and their induction was not affected by either TLR2 knockout or by TLR7 siRNA (Fig. 7A). IRF3 was constitutively expressed and not modulated either by TLR2 or by TLR7 activation (Fig. 7A). These results suggest that the phosphorylation of p65 NFB is largely dependent on TLR2, but the induction of KLF4 and IRF7 is independent of either TLR2 or TLR7. Because both MyD88 and IFNβR played important roles in gene induction by P. gingivalis, its LPS or its FimA (Fig. 5), we further analyzed the role of MyD88 and IFNβR in the regulation of p65 NFB, KLF4, IRF3, and IRF7 activation. As expected, the phosphorylation of p65 NFB via TLR2 was completely suppressed by MyD88 knockout, but was not affected by IFNβR siRNA. Both KLF4 and IRF7 induced by LPS were suppressed by IFNβR siRNA, but not by MyD88 knockout. The constitutively expressed IRF3 was also not affected by either MyD88 knockout or by IFNβR siRNA (Fig. 7B). Furthermore, in human macrophages, p65 NFB phosphorylation was also unimpaired by IFNβR siRNA; however, the induction of both KLF4 and IRF7 by P. gingivalis LPS was significantly diminished by IFNβR siRNA (Fig. 7C). Therefore, we can conclude that activation of p65 NFB by P. gingivalis, its LPS, or its FimA via TLR2 is dependent on MyD88, while activation of KLF4 and IRF7 by P. gingivalis LPS is dependent on IFNβR.

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Roles of TLR2, TLR7, MyD88, and IFN/βR in the activation of NFB, KLF4 and IRF7. A, Macrophages from WT and TLR2–/– mice were transfected with siRNA for TLR7, and treated with P. gingivalis, its LPS, or its FimA for 4 h. The cell lysates were analyzed by Western blotting. The data shown represent one of the three independent experiments. B, WT, MyD88–/–, and IFN/βR siRNA-treated macrophages were stimulated with P. gingivalis, its LPS, or its FimA for 4 h, and the cell lysates were analyzed by Western blotting. The data shown represent one of the three independent experiments. C, Human macrophages were transfected with IFN/βR siRNA and treated with P. gingivalis LPS for 2 and 4 h. The cell lysates were analyzed by Western blotting, and the data shown represent one of the three independent experiments.

Induction of pS727 STAT1 by P. gingivalis or its components is dependent on MyD88, while induction of pY701 STAT1 by LPS is dependent on IFNβR

Previous reports have demonstrated that both TLR-signaling and IFNβR-signaling lead to the phosphorylation of STAT1 (20, 26, 27). Subsequent studies sought to determine whether engagement of MyD88 and IFNβR also leads to the activation of STAT1 in response to the stimulation of P. gingivalis, its LPS, or its FimA. STAT1 activation was assessed by measuring its phosphorylation status. Our results demonstrated that P. gingivalis, its LPS, and its FimA all induced the serine phosphorylation of STAT1 (pS727) and that this induction was suppressed by MyD88 knockout, but not by IFNβR siRNA. However, the STAT1 tyrosine phosphorylation (pY701) was induced only by P. gingivalis LPS, and this phosphorylation was not affected by MyD88 knockout, but was severely impaired by IFNβR siRNA. Total levels of STAT1 did not change in response to each agonist, and no effect of MyD88 and IFNβR on total STAT1 was observed (Fig. 8). Thus, the serine phosphorylation of STAT1 induced by P. gingivalis, its LPS, or its FimA was dependent on MyD88, while the unique induction of the tyrosine phosphorylation of STAT1 by P. gingivalis LPS was dependent on IFNβR.

View larger version (19K): [in this window] [in a new window]   FIGURE 8. Effects of MyD88 and IFN/βR on STAT1 phosphorylation are shown. WT, MyD88–/–, and IFN/βR siRNA-treated macrophages were stimulated with P. gingivalis, its LPS, or its FimA for 4 h, and the phosphorylation of STAT1 was analyzed by Western blotting. The data shown represent one of the three independent experiments.

Because phosphorylation of kinases play important roles in signal transduction, we analyzed the phosphorylation status of all three major families of MAPKs, the ERK1/2, and cJNK1–3 by human MAPKs phosphorylation array. The results indicated that p38 MAPK, ERK1/ERK2, JNK2, and HSP27 were phosphorylated in human macrophages after treatment with P. gingivalis or its cell surface components (Fig. 9, A–C). Because p38 MAPK is identified as the most predominant phosphor-kinase (including p38,,) and because it has previously been shown to be necessary for TLR-induced phosphorylation of STAT1 (27), we further analyzed the role of p38 MAPK on gene induction of PTX3, CXCL10, IFN-β, IRF7, and IFIT2. To accomplish this, human macrophages were stimulated with P. gingivalis, its LPS, or its FimA, in the presence or absence (DMSO only) of the specific p38 inhibitor SB203580. Total RNA was analyzed by qRT-PCR for gene expression of PTX3, CXCL10, IFN-β, IRF7, and IFIT2. The results showed that SB203580 treatment inhibited PTX3 and CXCL10 induction by each agonist, but had little effect on IFN-β, IRF7, and IFIT2 induction by P. gingivalis LPS (Fig. 9, D–F). These findings demonstrated that the phosphorylation of p38 MAPK was necessary for P. gingivalis-, its LPS-, and its FimA-induced NFB-dependent gene expression, but not for P. gingivalis LPS-induced IFN-inducible gene expression.

View larger version (41K): [in this window] [in a new window]   FIGURE 9. p38 MAPK is essential for NFB-dependent gene induction by P. gingivalis, its LPS, and its FimA. Human macrophages were exposed to P. gingivalis, its LPS, or its FimA for 30 min, and cell lysates were subjected to phospho-MAPK array. The locations of each phospho-MAPK Ab in the array membrane are shown in A. B, The phospho-MAPK array images which represent one of three independent experiments obtained by exposure of membranes to x-ray film. Phosphorylated MAPK, whose average changes in net optical intensity (mean ± SD; n = 3) were greater than 2-fold in P. gingivalis-, LPS-, or FimA-treated cells relative to control cells, are shown in C. D–F, Human macrophages treated with DMSO or 20 µM SB203580 for 1 h before exposure to P. gingivalis, its LPS, or its FimA for 2 h. Gene expression of PTX3, CXCL10, IFNB1, IRF7, and IFIT2 were quantified by qRT-PCR using total cellular RNA. The data shown is the average fold changes of three independent experiments. Asterisks indicate statistically significant (p < 0.05) differences in change of mRNA expression.

	Discussion

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In the present study, we demonstrated that P. gingivalis, its LPS, and its FimA commonly activate NFB through either TLR2- or TLR7-MyD88-p38 MAPK pathway to induce NFB-containing and NFB/IRSE cocontaining gene expression, while P. gingivalis LPS uniquely induces ISRE-containing gene by the activation of IFN-β autocrine loop. Although endowed with critical antiviral activities, type I IFN signaling plays uncertain roles in antibacterial defenses. It had only minor effects on the outcome of experimental lung tuberculosis (36), but detrimental in listeriosis (37, 38, 39); whereas it was a crucial host defense against extracellular patho-gens, including group B Streptococci pneumonia, E. coli, and Sal-monella typhimurium (40). IFN-β signaling was required for optimal macrophage responses to low dose of purified LPS, which may mimic the actual amount of LPS in the infection tissue, to increase the TNF- and IFN- production in macrophages (40). Therefore, P. gingivalis LPS possibly could boost the antibacterial responses of macrophages by promoting IFN-β expression, and simultaneously cause detrimental lesion in infected tissues due to enhanced local inflammatory reactions, such as overexpression of TNF-.

P. gingivalis is also strongly associated with the development of atherosclerosis (28, 29, 30, 31, 32, 33); this may be attributed to its ability to activate TLR2 given that TLR2 plays a critical role in the progression of atherosclerosis (41, 42, 43, 44). During P. gingivalis chronic infection, the bacteria themselves and their LPS and fimbriae released via bacterial breakdown might penetrate the gingival tissues and move into blood circulation, from which they might contribute to systemic inflammatory responses, such as those believed to be involved in atherosclerosis. P. gingivalis LPS and FimA have been demonstrated to have the ability to induce the production of IL-8 and MCP-1 in human vascular endothelial cells (45) and macrophages (9), which is closely related to atherosclerosis (46, 47). TLR2 activation by P. gingivalis LPS and fimbriae in the blood circulation system may also impart macrophage-activating ability to apolipoprotein A-1 (44), therefore, reverse the protective role of apoA-1 and contribute to the genesis of atherosclerosis. Finally DPG3, a fimbriae-deficient strain of P. gingivalis was not able to stimulate atherosclerosis as did the wild-type strain advocating for a role of fimbriae in P. gingivalis-associated atherosclerosis (45, 48, 49, 50).

In addition, our studies demonstrated that TLR7 was activated by P. gingivalis, its LPS, or its FimA, and it played an important role in the induction of NFB-containing gene as well as NFB-ISRE cocontaining gene expression. The study by Triantafilou et al. (28) showed that the silencing of TLR7 did not affect the production of TNF- in human vascular endothelial cells in response to P. gingivalis LPS. However, in our study we used macrophages, a cell type that is different from endothelial cells, which can explain the discrepancy. In cooperation with TLR4 signaling, TLR7/8 was shown recently to induce IL-12p70 synthesis in human monocytes, triggering a potent Th1 response before T cell help is established (51). However, consistent with our results that TLR2 had little effect on the early induction of IFN-β, a potential IL-12p70 inducer (20), the combination of TLR2 and TLR7/8 signaling was found unable to induce IL-12p70 in human monocytes (51). Interestingly, we observed that TLR7 was essential for the early induction of IFN-β (Fig. 4) by P. gingivalis LPS, and this induction required an intact IFNβR signaling. This double requirement for IFN-β induction may represent a safeguard mechanism preventing inappropriate secretion of potentially harmful Th1 cytokines induced by type I IFN in the early phase of an infection. The mechanism involved in the initial IFN-β induction by TLR7 may require the formation of a complex consisting of MyD88, IRF7, and TRAF6 (52, 53). Although both TLR2 and TLR7 activate MyD88 pathway, TLR7 uses only MyD88 as an adaptor to transmit a signal. In contrast, TLR2 uses both MyD88 and TIRAP (54). Thus, it is possible that the newly induced IRF7 by P. gingivalis LPS or its associated protein kinase cannot be recruited to the TLR-MyD88 complex when TIRAP is associated with the receptor complex or that TIRAP mediates an inhibitory signal that interferes with IRF7 phosphorylation. We also found that the induction of IRF7 requires an IFNβR signaling. This may explain why the initial induction of IFN-β requires signal from both TLR7 and IFNβR. Our results show that the early weak induction of IFN-inducible genes (IFN-β and IFIT2) by P. gingivalis LPS was significantly suppressed by siRNA for IFN/βR. Therefore, P. gingivalis LPS may be a weak activator for IFN/βR and induces a minimal initial IFN-β expression because it induces the tyrosine phosphorylation of STAT1 (Fig. 8) and IRF7 expression (Fig. 5C). After a certain amount of IFN-β has been produced, the robust expression of IFN-inducible genes is then induced by IFN-β autocrine loop. Nevertheless, the initial activator of IFNβR requires further investigation. Further studies will examine whether P. gingivalis LPS can directly activate and bind to the IFN/βR.

Based on our present data, we postulated a signaling pathway model induced by P.gingivalis, its LPS and its FimA (Fig. 10). P. gingivalis, its LPS and its FimA activate either TLR2 or TLR7 and induce NFB-containing or NFB/IFNβR cocontaining genes through MyD88-p38 MAPK pathway in connection with the use of STAT1 (pS727)/p65 complexes or KLF/p65 complexes. P. gingivalis LPS additionally induces expression of ISRE-containing genes requiring the activation of IFNβR-STAT1 (pY701) pathway in connection with the use of p65/IRF7 or KLF4/IRF7 complexes. Because IRF7 is absolutely required for robust IFN/β induction (23, 55), the maximum induction of IFN-β by P. gingivalis LPS may require the activation of both IRF7 and p65 NFB. Because the induction of KLF4 and IRF7 by P. gingivalis LPS was dependent on IFNβR, and the promoters of ISRE-containing genes possess both ISRE and KLF-binding sites; therefore, KLF4 together with IRF7 may play an important role in the induction of ISRE-containing genes, such as IFIT2. In summary, we have identified these signaling pathways based on a broad range of information obtained in this study. Although the TLR-MyD88-p38 MAPK pathway is well-known for macrophage reaction to pathogens, the IFNβR signaling pathway could also be of major importance in determining the nature of the course of infection and pathology (i.e., acute vs chronic) in P. gingivalis infection because P. gingivalis LPS is a potent inductor of IFN-β. Determining the physiological role of type I IFNs during P. gingivalis infection will be the next critical step in understanding the chronic inflammatory reaction of periodontitis caused by P. gingivalis.

View larger version (23K): [in this window] [in a new window]   FIGURE 10. Model of signaling pathways of macrophage initiated by P. gingivalis (PG), its LPS, and its FimA. Both P. gingivalis and its purified components activate TLR2 and TLR7 to induce NFB-dependent gene expression through MyD88-p38 MAPK pathway. In addition, P. gingivalis LPS induces ISRE-containing gene expression requiring IFN/βR signal from IFN-β, which leads to the tyrosine phosphorylation of STAT1 and the activation of IRF7 and KLF4.

	Disclosures

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	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the National Institute of Dental and Craniofacial Research DE15989 (to S.A.).

² Address correspondence and reprint requests to Dr. Salomon Amar, Department of Periodontology and Oral Biology, School of Dental Medicine, Boston University Medical Center, 700 Albany Street, W-201E, Boston, MA 02118. E-mail address: samar{at}bu.edu

³ Abbreviations used in this paper: FimA, fimbrial protein; ISRE, IFN-sensitive response element; MOI, multiplicity of infection; TF, transcription factor; qRT-PCR, quantitative real-time PCR; RT, reverse transcription; C_T, cycle threshold; KLF, Kruppel-like factor.

Received for publication November 7, 2006. Accepted for publication September 26, 2007.

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