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The Journal of Immunology, 2007, 179, 2359 -2367
Copyright © 2007 by The American Association of Immunologists, Inc.
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Complement Receptor 3 Blockade Promotes IL-12-Mediated Clearance of Porphyromonas gingivalis and Negates Its Virulence In Vivo1

George Hajishengallis2,*,{dagger}, Muhamad-Ali K. Shakhatreh*, Min Wang* and Shuang Liang*

* Division of Oral Health and Systemic Disease/Department of Periodontics, and {dagger} Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ability of certain pathogens to exploit innate immune function allows them to undermine immune clearance and thereby increase their persistence and capacity to cause disease. Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with increased risk of systemic conditions. We have previously shown that the fimbriae of P. gingivalis interact with complement receptor 3 (CR3; CD11b/CD18) in monocytes/macrophages, resulting in inhibition of IL-12p70 production in vitro. The in vivo biological implications of this observation were investigated in this study using a CR3 antagonist (XVA143). XVA143 was shown to block CR3 binding of P. gingivalis fimbriae and reverse IL-12p70 inhibition; specifically, CR3 blockade resulted in inhibition of ERK1/2 phosphorylation and up-regulation of IL-12 p35 and p40 mRNA expression. Importantly, mice pretreated with XVA143 elicited higher IL-12p70 and IFN-{gamma} levels in response to P. gingivalis i.p. infection and displayed enhanced pathogen clearance, compared with similarly infected controls. The notion that CR3 is associated with reduced IL-12p70 induction and impaired P. gingivalis clearance was confirmed using i.p. infected wild-type and CR3-deficient mice. Moreover, XVA143 dramatically attenuated the persistence and virulence of P. gingivalis in experimental mouse periodontitis, as evidenced by reduced induction of periodontal bone loss. Therefore, CR3 blockade may represent a promising immunomodulatory approach for controlling human periodontitis and possibly associated systemic diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The beta2 integrins are heterodimeric receptors consisting of a common beta subunit (CD18) associated noncovalently with a unique {alpha} subunit (CD11a, b, c, or d) (1). These receptors orchestrate crucial processes involved in immunity and inflammation and, in this context, mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions (2, 3). To integrate the intracellular and extracellular environments, beta2 integrins use inside-out and outside-in bidirectional signaling. Inside-out signaling refers to dynamic regulation of their adhesive activity from within the cell via signals generated by other receptors, whereas outside-in signaling refers to the ability of the activated integrin to respond to ligands by inducing downstream intracellular signaling (1). CD11b/CD18 is also known as complement receptor 3 (CR3)3 and is abundantly expressed by phagocytic leukocytes (2). CR3 has the capacity to interact with a wide variety of structurally unrelated molecules derived from either the host (e.g., ICAM-1, fibrinogen, and the complement fragment iC3b) or pathogens (e.g., Bordetella pertussis filamentous hemagglutinin and Leishmania gp63) (4, 5, 6). The ligand-binding promiscuity of CR3 suggests that it may function as a pattern-recognition receptor (PRR) and, in this regard, CR3 clusters with other PRRs (e.g., CD14 and TLRs) in lipid rafts of activated cells (7, 8).

We have recently described a novel inside-out signaling pathway in human monocytes or mouse macrophages activated by Porphyromonas gingivalis (9, 10), an oral pathogen that is strongly associated with periodontal disease and implicated in atherosclerosis (11, 12). This proadhesive pathway is initiated when P. gingivalis fimbriae interact with the CD14/TLR2 recognition/signaling complex, leading to PI3K-mediated induction of the high-affinity conformation of CR3 (9, 10, 13). Activation of this TLR2 inside-out signaling pathway by P. gingivalis or purified fimbriae leads to enhanced CR3-dependent monocyte adhesion and transendothelial migration (10). However, additional work by our group has shown that P. gingivalis has co-opted this proadhesive pathway for CR3 binding, intracellular entry, and inhibition of IL-12p70 (13, 14, 15) (Fig. 1A), a heterodimeric cytokine composed of disulfide-linked p35 and p40 subunits (16). We have thus speculated that CR3 may be exploited by P. gingivalis for undermining IL-12-mediated bacterial clearance, thereby promoting its virulence and capacity to cause disease. Interestingly, several pathogens have developed distinct mechanisms for IL-12 suppression (5, 17, 18, 19, 20). This may represent an effective microbial strategy for immune evasion because production of IL-12p70 by macrophages is a key event in host defense against infection (16). Indeed, IL-12p70 activates T and NK cells to produce IFN-{gamma}, which in turn activates the bactericidal function of macrophages (16).


Figure 1
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  		FIGURE 1. P. gingivalis colocalizes with TLR2 and CR3 and exploits these interactions to undermine innate immunity. A, P. gingivalis exploits its fimbrial-mediated interaction with macrophage TLR2 to activate the high-affinity conformation of CR3 within minutes. This pathway proceeds through Rac1/PI3K-mediated inside-out signaling and requires CD14 for facilitating the fimbria-TLR2 interaction (9 10 ). TLR2 stimulation by P. gingivalis also induces NF-{kappa}B activation and cytokine production (8 14 ). Upon CR3 activation, fimbriated P. gingivalis can readily interact with CR3, leading to outside-in signaling, involving ERK1/2, which down-regulates IL-12 p35 and p40 and consequently inhibits IL-12p70 production (this study). In vivo evidence presented in this study indicates that exploitation of CR3 function by P. gingivalis undermines IL-12-mediated immune clearance, leading to persistence of the pathogen and enhanced virulence in the mouse periodontitis model. B, Mouse macrophages were exposed to FITC-labeled P. gingivalis for 20 min. After cell fixation and staining with allophycocyanin-labeled anti-TLR2 mAb and Texas Red-labeled anti-CD11b mAb, the cells were examined by confocal microscopy. The merge images demonstrate colocalization of P. gingivalis with both TLR2 and CR3.

 
Because macrophages play important roles at the front line of host defense while at the same time constituting targets of pathogens aiming to subvert immune surveillance (21), the elucidation of macrophage-pathogen interactions is essential to understanding the pathogenesis of many infection-driven diseases, including periodontitis. The notion that macrophage CR3 function may be exploited by P. gingivalis for undermining IL-12-mediated bacterial clearance seems paradoxical at first, given that CR3 can potentially play important roles in innate immune defense against microbial pathogens (2). Nevertheless, at least certain pathogens appear to promote their survival in the host by using CR3 (22, 23, 24, 25), which, unlike other phagocytic receptors, is not linked to vigorous microbicidal mechanisms (21, 26, 27, 28, 29).

On the basis of our in vitro findings outlined above, we hypothesized that CR3 blockade in vivo may counteract an important immune evasion mechanism of P. gingivalis and thereby provide host protection. Indeed, we now present evidence that the in vivo use of a CR3 antagonist reverses the ability of P. gingivalis to suppress induction of IL-12p70 and IFN-{gamma}, thereby leading to enhanced clearance of this pathogen from systemically infected mice. Importantly, the use of the CR3 antagonist moreover protects orally infected mice against P. gingivalis-induced periodontitis, as evidenced by reduced induction of periodontal bone loss. Because the ability of P. gingivalis to proactively suppress innate responses is likely to compromise the capacity of the host to clear also other bacteria that cohabit its niche, our findings hold promise for the use of CR3 antagonists in controlling human periodontitis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

P. gingivalis 33277 and its FimA-deficient isogenic mutant JI-1 (donated by F. Yoshimura, Aichi-Gakuin University, Nagoya, Japan) were grown anaerobically at 37°C in modified GAM medium (contains 5 µg/ml hemin and 1 µg/ml menadione) (Nissui Pharmaceutical). Fimbriae were purified, as previously described (9). The fimbrial preparations were free of any contaminating substances on silver-stained SDS-PAGE, and tested negative for endotoxin (<6 EU/mg protein), according to quantitative Limulus amebocyte lysate assay (BioWhittaker). Escherichia coli K-12 LPS was purchased from InvivoGen. Aggregatibacter (Actinomyces) actinomycetemcomitans LPS was provided by E. Haase (University of Buffalo, Buffalo, NY) (30). The small-molecule inhibitor XVA143 (m.w. 585.35), which blocks CR3 (10, 31), was provided by N. Fotouhi (Roche, Nutley, NJ). Recombinant human or mouse IFN-{gamma}, goat polyclonal anti-mouse IL-12 IgG or anti-mouse IL-23 (p19) IgG, and an IgG2b mAb to human IL-10 were purchased from R&D Systems. PD98059 was from Cell Signaling Technology. The reagents were used at effective concentrations determined in preliminary experiments or in previous publications.

Cell culture

Monocytes were purified from human peripheral blood upon centrifugation over NycoPrep 1.068 (Axis-Shield) (9). Incidental nonmonocytes were removed by magnetic depletion using a mixture of biotin-conjugated mAbs and magnetic microbeads coupled to anti-biotin mAb (Monocyte Isolation Kit II; Miltenyi Biotec). Purified monocytes were cultured at 37°C and 5% CO2 atmosphere, in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.05 mM 2-ME (complete RPMI 1640). Mouse macrophages were isolated from the peritoneal cavity upon thioglycolate-induced elicitation (32) and were cultured in complete RPMI 1640. Human blood collections and isolation of mouse macrophages were conducted in compliance with established federal guidelines and institutional policies. Chinese hamster ovary (CHO) cells stably transfected with human CR1 or CR3 were provided by D. Golenbock (University of Massachusetts Medical School, Worcester, MA) (33). CHO cells were cultured in Ham’s F-12 nutrient mixture (Invitrogen Life Technologies) supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cell viability was monitored using the CellTiter-Blue assay kit (Promega). None of the experimental treatments affected cell viability compared with medium-only control treatments.

Fimbria-binding assay

The binding of purified fimbriae to CHO/CR3 cells was performed using a fluorescent cell-based assay in 96-well plates, as we previously described (13). Briefly, biotinylated fimbriae (1 µg/ml) were allowed to bind for 30 min at 37°C, and bound protein was probed with FITC-labeled streptavidin. Upon washing, binding was determined by measuring cell-associated fluorescence on a microplate fluorescence reader (FL600; Bio-Tek Instruments) with excitation/emission wavelength settings of 485/530 nm. Because CHO/CR3 cells do not express functional TLR2 and thus CR3 cannot be activated by fimbria-induced TLR2 inside-out signaling (13), PMA (0.1 µg/ml) was used to activate CR3 in the binding assay.

Cell activation assays

Human monocytes or mouse macrophages were stimulated with P. gingivalis fimbriae or E. coli LPS, as outlined in the figure legends, and induction of cytokine release (TNF-{alpha}, IL-1beta, IL-6, IL-8, and IL-12p70) in culture supernatants was measured by ELISA using kits from eBioscience or Cell Sciences. Total and phosphorylated ERK1/2 was determined using total ERK1/2 and PhosphoDetect ERK1/2 (pTpY185/187) ELISA kits from Calbiochem.

Real-time PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen Life Technologies) and quantified by spectrometry at 260 and 280 nm. First strand cDNA synthesis was performed using the High-Capacity cDNA Archive kit (Applied Biosystems). Real-time PCR with cDNA was performed with an ABI 7500 system (Applied Biosystems). TaqMan probes, sense primers, and antisense primers for gene expression of human IL-12p35 and IL-12p40 or a housekeeping gene (GAPDH) were purchased from Applied Biosystems. Using a Universal PCR Master Mix (Applied Biosystems), the reactions were performed, according to the manufacturer’s protocol. Relative expression level was determined by normalization to the housekeeping gene.

Confocal microscopy

To demonstrate colocalization of P. gingivalis with TLR2 and CR3, mouse macrophages were grown on chamber slides (Lab-Tek) and were exposed to FITC-labeled P. gingivalis for 20 min. Immediately afterward, the cells were fixed with 2% paraformaldehyde and permeabilized with.05% Triton X-100. Cells were then stained with Texas Red-labeled anti-CD11b mAb (3A33; Abcam) and allophycocyanin-labeled anti-TLR2 mAb (6C2; eBioscience) and mounted with coverslips before imaging on an Olympus FV500 confocal microscope.

In vivo mouse studies

Peritonitis model. Specific pathogen-free BALB/cByJ mice (8–10 wk old; The Jackson Laboratory) were pretreated by i.p. injection of XVA143 (0.5 ml of 50 µM) or PBS alone. After 1 h, the mice were infected i.p. with P. gingivalis 33277 (5 x 107 CFU). Peritoneal lavage was performed 20 h postinfection. Serial 10-fold dilutions of peritoneal fluid were plated onto blood agar plates supplemented with hemin/menadione and cultured anaerobically for enumerating recovered peritoneal CFU. The peritoneal fluid was also used to measure IL-12p70 and IFN-{gamma} production by ELISA. In a similar, but modified experiment, XVA143-treated and P. gingivalis-challenged mice were previously given i.p. goat polyclonal anti-mouse IL-12 IgG (0.1 mg/mouse) or equal amount of nonimmune IgG. Similar procedures were followed to determine P. gingivalis clearance from the peritoneal cavity of infected wild-type or CR3-deficient (CD11b–/–) C57BL/6 mice (The Jackson Laboratory).

Periodontitis model. Ten-week-old BALB/cByJ mice were orally infected with P. gingivalis American Type Culture Collection 33277 for induction of periodontal bone loss, according to the Baker et al. model (34). To determine the role of CR3 blockade on P. gingivalis-induced bone loss, the mice were administered XVA143 or PBS control through microinjection. Specifically, XVA143 was injected using a 28.5-gauge MicroFine needle (BD Biosciences) into the palatal gingivae, on the mesial of the first molar and in the papillae between first and second and third molars on both sides of the maxilla (1 µl of 1 mM solution per site; total of six sites). XVA143 was administered five times at 2-day intervals (days 1, 3, 5, 7, and 9). Each administration of XVA143 or PBS preceded, by 1 day, oral infection with 109 CFU P. gingivalis 33277 in 2% carboxymethylcellulose or with carboxymethylcellulose vehicle alone (sham infection), also given five times at 2-day intervals (i.e., days 2, 4, 6, 8, and 10). Therefore, four groups were used (PBS pretreated/sham infected; PBS pretreated/P. gingivalis infected; XVA143 pretreated/sham infected; and XVA143 pretreated/P. gingivalis infected), each comprising five mice. Six weeks after the final infection, mice were euthanized with CO2 inhalation. Assessment of periodontal bone loss in defleshed maxillae was performed under a dissecting microscope (x40) fitted with a video image marker measurement system (model VIA-170K; Fryer) standardized for measurements in millimeters (mm). Specifically, the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured on 14 predetermined points on the palatal surfaces of the maxillary molars (34). To calculate periodontal bone loss, the 14-site total CEJ-ABC distance for each mouse was subtracted from the mean CEJ-ABC distance from the group of sham-infected mice; results were expressed as mm change in bone, and negative values indicated bone loss compared with sham-infected controls (34). All animal procedures received Institutional Animal Care and Use Committee approval and were in accordance with federal guidelines for the care and use of laboratory animals.

Statistical analysis

Data were evaluated by ANOVA and the Dunnett multiple-comparison test using the InStat program (GraphPad). Where appropriate (comparison of two groups only), two-tailed t tests were also performed. Statistical differences were considered significant at the level of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Inhibition of P. gingivalis fimbria-CR3 interaction and up-regulation of IL-12p70 by a CR3 antagonist

P. gingivalis fimbriae stimulate TLR2 inside-out signaling for activation and binding of CR3 (CD11b/CD18) (9, 13), which is consistent with the observation that this pathogen colocalizes with both PRRs in macrophages (Fig. 1B). To determine the biological implications of CR3 binding by P. gingivalis fimbriae, we used XVA143 (m.w. 585.35), an allosteric antagonist that binds CD18 and blocks transmission of activation signals to the ligand-binding domain of CD11b (35). Although we have previously shown that XVA143 blocks the binding activity of human or mouse CR3 for ICAM-1 or fibrinogen (10), it was essential to determine whether XVA143 can similarly inhibit CR3 binding of P. gingivalis fimbriae. The fimbriae could readily bind CR3-expressing, but not CR1-expressing CHO cells; however, XVA143 inhibited binding by >80% (p < 0.05) and its effect was comparable to a blocking anti-CD11b mAb (Fig. 2). We next examined the ability of XVA143 to antagonize CR3-dependent biological activities of P. gingivalis fimbriae. Because P. gingivalis fimbriae inhibit production of IL-12p70 by interacting with CR3, we have investigated the ability of XVA143 to reverse this potential immune evasion effect. For this purpose, human monocytes were stimulated with fimbriae (1 µg/ml) in the absence or presence of XVA143 (1 µM). XVA143 partially blocked fimbria-induced TNF-{alpha}, IL-1beta, IL-6, or IL-8 (p < 0.05; Fig. 3, A–D), but exerted an up-regulating effect on IL-12p70 production (Fig. 3E). In a separate experiment in which monocytes were previously primed with IFN-{gamma} (0.1 µg/ml) to enhance IL-12p70 induction, XVA143 maintained the ability to further up-regulate fimbria-induced IL-12p70 production by >5-fold (p < 0.05; Fig. 3F). Similar results were obtained using mouse macrophages (data not shown). These findings suggest that XVA143 reverses the inhibitory effect of the P. gingivalis fimbria-CR3 interaction on IL-12p70 induction.


Figure 2
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  		FIGURE 2. XVA143 blocks CR3-dependent binding of P. gingivalis fimbriae. CHO cells stably transfected with CR1 or CR3 were incubated with biotinylated P. gingivalis fimbriae (1 µg/ml) for 30 min at 37°C. The binding of fimbriae was measured as cell-associated fluorescence after cell staining with streptavidin (SA)-FITC. Background binding was determined in cells treated with medium only and SA-FITC. The binding of fimbriae was assessed in the absence or presence of anti-CD11b or IgG1 isotype control (both at 10 µg/ml) or XVA143 (1 µM). The horizontal discontinuous line indicates low-level, CR3-independent binding. Data are presented as means ± SDs (n = 3) from one of two independent experiments that yielded similar results. Asterisks denote statistically significant (p < 0.05) inhibition of binding. RFU, relative fluorescence units.

 

Figure 3
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  		FIGURE 3. CR3 blockade in human monocytes inhibits release of several cytokines, but enhances IL-12p70 in response to P. gingivalis fimbriae. Human monocytes were pretreated with or without XVA143 (1 µM) for 30 min and then incubated with medium only or fimbriae (1 µg/ml). After 16 h, supernatants were collected for cytokine analysis by ELISA (A–E). F, Shows a separate experiment for determining IL-12p70 induction using IFN-{gamma}-primed monocytes. Results are shown as means ± SD (n = 3) from one of two independent sets of experiments that yielded similar findings. Asterisks indicate significant (p < 0.05) differences in cytokine induction due to treatment with XVA143.

 
The P. gingivalis fimbria-CR3 interaction down-regulates IL-12 p35 and p40 through ERK1/2 signaling

We next investigated the effect of XVA143 on expression of the individual IL-12 p35 and p40 subunits at the mRNA level. Using real-time PCR and XVA143-pretreated monocytes exposed to P. gingivalis fimbriae, we found that XVA143 enhanced induction of both IL-12 p35 and p40 subunits compared with XVA143-nonpretreated (PBS) controls (p < 0.05; Fig. 4, A and B). Because IL-12 is controlled by ERK1/2 signaling in several experimental systems (36, 37), we next determined the IL-12 regulatory effect of PD98059 (20 µM), which inhibits the activation (phosphorylation) of ERK1/2 (36). Similarly to XVA143, PD98059 exerted a potent up-regulating effect on the expression of both IL-12 p35 and p40 mRNA and on the production of IL-12p70 protein (p < 0.05 compared with 0.1% DMSO vehicle control; Fig. 4, C–E). In contrast, pretreatment with a neutralizing dose of anti-IL-10 mAb resulted in modest up-regulation of IL-12p70, at levels <30% of those seen with PD98059 (Fig. 4F). This suggests that IL-12 down-regulation by the P. gingivalis fimbria-CR3 interaction is only partially attributable to endogenously produced IL-10. We next showed that ERK1/2 is activated by P. gingivalis fimbriae in a CR3-dependent way. Indeed, fimbriae induced significant phosphorylation of ERK1/2, which was reversible by CR3 blockade using XVA143 (p < 0.05; Fig. 4G). None of the treatments influenced total ERK1/2 levels (Fig. 4G). The Fig. 4 data collectively suggest that the interaction of P. gingivalis fimbriae with CR3 induces ERK1/2 phosphorylation leading to down-regulation of IL-12 p35 and p40 subunits, and suppression of bioactive (p70) IL-12 production.


Figure 4
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  		FIGURE 4. The interaction of P. gingivalis fimbriae with CR3 inhibits IL-12 expression through ERK1/2. Human monocytes were pretreated or not for 45 min with 1 µM XVA143 (A and B) or with 20 µM PD98059 (C and D), and subsequently incubated for 5 h with medium only or with P. gingivalis fimbriae. Real-time PCR was used to determine IL-12 p35 and p40 mRNA levels, normalized against GAPDH mRNA levels. Data are shown as fold induction relative to medium-only treated control. E and F, PD98059-pretreated monocytes were incubated with fimbriae for 16 h, with or without IFN-{gamma} (0.1 µg/ml), and culture supernatants were assayed for IL-12p70. F, The IL-12 up-regulatory effect of a neutralizing anti-IL-10 mAb (10 µg/ml) or IgG2b isotype control (IC) was compared with that of PD98059. G, Cells were pretreated with XVA143, and fimbria-induced phosphorylation of ERK1/2 was determined using PhosphoDetect ERK1/2 (pTpY185/187) ELISA. Total ERK1/2 content was quantitated using Total ERK1/2 ELISA kit. Results are means ± SD (n = 3) and were confirmed in repeated experiments. Asterisks indicate significant (p < 0.05) differences between experimental and control treatments.

 
Enhanced in vivo clearance of P. gingivalis by CR3 antagonist

IL-12p70 production by macrophages activates T cells and NK cells to produce IFN-{gamma}, which in turn activates the bactericidal function of macrophages (16). We have thus investigated the effect of IL-12p70 induction on the in vivo clearance of P. gingivalis. The CR3 antagonist XVA143, which up-regulates IL-12 in vitro (Fig. 3, E and F, and Fig. 4, A and B), was used as the immunomodulatory agent. Initially, we tested the ability of XVA143 to up-regulate IL-12p70 in vivo. Mice were administered XVA143 or PBS by the i.p. route, followed 1 h later with i.v. challenge with P. gingivalis 33277. XVA143 pretreatment of P. gingivalis-challenged mice resulted in significantly higher serum levels of IL-12p70 and IFN-{gamma} compared with pretreatment with PBS vehicle control (p < 0.05; Fig. 5). No IL-12p70 or IFN-{gamma} was detectable in mice not challenged with P. gingivalis regardless of whether they had previously received XVA143 or not (Fig. 5).


Figure 5
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  		FIGURE 5. The CR3 antagonist, XVA143, enhances in vivo IL-12p70 and IFN-{gamma} production. BALB/cByJ mice were pretreated with XVA143 (i.p.; 0.5 ml of 50 µM) or PBS alone. After 1 h, the mice were i.v. injected with P. gingivalis 33277 (5 x 107 CFU) or PBS alone. Six hours later, blood was collected to analyze serum levels of IL-12p70 and IFN-{gamma}. Results are means ± SD (n = 4) from one of two experiments yielding similar findings. Asterisks indicate significant (p < 0.05) differences in cytokine production due to pretreatment with XVA143.

 
We then determined whether XVA143 helps the mice clear an i.p. infection with P. gingivalis. We found that 20 h postinfection, the peritoneal lavage fluid from XVA143-pretreated mice contained ~40 times less P. gingivalis CFU compared with mice pretreated with PBS instead (p < 0.05; Fig. 6A). Analysis of peritoneal fluid for IL-12p70 and IFN-{gamma} confirmed the enhancing effect of XVA143 on the production of these cytokines (Fig. 6B). Therefore, positive regulation of IL-12p70 and IFN-{gamma} by XVA143 correlates with enhanced in vivo clearance of P. gingivalis. To conclusively implicate IL-12p70 in protection, mice were i.p. given goat polyclonal anti-mouse IL-12 IgG (0.1 mg/mouse; R&D Systems) or an equal amount of nonimmune IgG. Both groups were then administered XVA143 and 1 h later were challenged with P. gingivalis. Mice treated with anti-IL-12 yielded significantly higher levels of P. gingivalis CFU from the peritoneal cavity 20 h postinfection, compared with mice receiving nonimmune IgG (p < 0.05; Fig. 6C). The anti-IL-12 Ab used reacts with both IL-12 p35 and p40, which is shared by IL-23, a heterodimeric cytokine containing a shared p40 and a unique p19 subunit (38). It was thus possible that the observed P. gingivalis clearance was mediated, at least in part, by IL-23. However, the use of goat anti-mouse IL-23 p19-neutralizing Ab had no significant effect on P. gingivalis clearance (Fig. 6C), suggesting that clearance is attributable to IL-12p70.


Figure 6
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  		FIGURE 6. The CR3 antagonist, XVA143, enhances the in vivo clearance of P. gingivalis by augmenting IL-12 and IFN-{gamma}. BALB/cByJ mice were pretreated by i.p. injection of XVA143 (0.5 ml of 50 µM) or PBS alone. After 1 h, the mice were infected i.p. with P. gingivalis 33277 (5 x 107 CFU). Peritoneal lavage was performed 20 h postinfection. Serial 10-fold dilutions of peritoneal fluid were plated for anaerobic growth and enumeration of recovered peritoneal CFU (A). The peritoneal fluid was also used to measure IL-12p70 and IFN-{gamma} production (B). C, Shows a similar experiment, except that XVA143-treated/P. gingivalis-challenged mice were previously given i.p. goat polyclonal anti-mouse IL-12 IgG or anti-mouse IL-23 p19 IgG (0.1 mg/mouse) or equal amount of nonimmune IgG. Data are shown for each mouse, and horizontal lines indicate the mean value (A and C) or are shown as means ± SD (n = 5) (B), from one of two sets of experiments that yielded similar results. Asterisks indicate significant (p < 0.05) differences in cytokine production (B) or in P. gingivalis peritoneal CFU (A and C) compared with control treatments.

 
P. gingivalis fimbriae inhibit LPS-induced IL-12p70 induction in vitro and in vivo

The ability of P. gingivalis fimbriae to suppress IL-12p70 induction through CR3 interaction has prompted us to investigate whether fimbriae can exert a similar inhibitory effect in cells activated with potent inducers of IL-12p70. For this purpose, we used IFN-{gamma}-primed and E. coli LPS-activated human monocytes. We found that the ability of LPS to induce IL-12p70 in monocytes was significantly inhibited in the presence of fimbriae, although an enhancing additive effect was observed for four other proinflammatory cytokines (p < 0.05; Fig. 7). Similar results were obtained using LPS purified from the oral pathogen A. actinomycetemcomitans (data not shown). Moreover, mice systemically challenged with E. coli LPS elicited significantly less serum IL-12p70 or IFN-{gamma} if they were previously administered P. gingivalis fimbriae rather than PBS control (p < 0.05; Fig. 8). Therefore, P. gingivalis fimbriae interfere with LPS-induced IL-12p70 production in vivo as seen in vitro.


Figure 7
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  		FIGURE 7. P. gingivalis fimbriae specifically inhibit IL-12p70 induction by E. coli LPS in monocytes. Human monocytes were primed with IFN-{gamma} (0.1 µg/ml) and stimulated with medium only (M), E. coli LPS (0.2 µg/ml), P. gingivalis fimbriae (F; 1 µg/ml), or both LPS and fimbriae. After 16 h, culture supernatants were assayed for induction of the indicated cytokines (A–E). Results are shown as means ± SD (n = 3) from a typical experiment that was performed three times yielding similar findings. Asterisks indicate statistically significant (p < 0.05) increase (A–D) or inhibition (E) of cytokine induction in cells exposed to LPS plus fimbriae compared with cells exposed to LPS only.

 

Figure 8
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  		FIGURE 8. P. gingivalis fimbriae inhibit E. coli LPS-induced IL-12p70 and IFN-{gamma} production in vivo. BALB/cByJ mice were pretreated with P. gingivalis fimbriae (i.p.; 10 µg) or PBS alone. After 1 h, the mice were i.v. injected with E. coli LPS (1 µg). Six hours later, blood was collected to analyze serum levels of IL-12p70 and IFN-{gamma}. Data are means ± SD (n = 4) from one of two experiments yielding similar results. Asterisks indicate significant (p < 0.05) differences in cytokine production due to pretreatment with fimbriae.

 
Enhanced in vivo clearance of P. gingivalis in CR3-deficient mice

The findings that XVA143 promotes P. gingivalis clearance (Fig. 6) are attributable to CR3 blockade by XVA143 and the consequent inability of P. gingivalis to exploit CR3 for enhancing its survival. This is a significant finding because XVA143 may be a useful immunomodulatory agent for controlling P. gingivalis infection. However, to conclusively implicate CR3 as an exploited receptor, we examined the ability of P. gingivalis 33277 to survive in i.p. infected wild-type or CR3-deficient mice. We found that 20 h postinfection, the peritoneal lavage fluid from CR3-deficient mice contained significantly lower P. gingivalis CFU, but significantly higher IL-12p70 and IFN-{gamma} levels, compared with wild-type mice (p < 0.05; Fig. 9, A and B). Therefore, the presence of CR3 correlates with reduced induction of IL-12p70 and IFN-{gamma} and impaired pathogen clearance. In a similar experiment using a nonfimbriated isogenic mutant (JI-1), we did not observe significant differences in the number of recovered CFU or the levels of IL-12p70 and IFN-{gamma} between wild-type and CR3-deficient mice (Fig. 9, C and D). In the absence of fimbriae, therefore, P. gingivalis cannot effectively exploit CR3 for inhibiting production of IL-12p70/IFN-{gamma} and increasing its survival potential. Consistent with this, the recovery of viable JI-1 CFU from wild-type mice was ~10-fold lower compared with the recovery of viable 33277 CFU; moreover, in CR3-deficient mice the wild-type strain (33277) lost its enhanced survival potential compared with the mutant (Fig. 9, A and C).


Figure 9
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  		FIGURE 9. Effect of CR3 deficiency on in vivo clearance of P. gingivalis and induction of IL-12 and IFN-{gamma}. Wild-type or CR3-deficient (CD11b–/–) C57BL/6 mice were infected i.p. with wild-type P. gingivalis 33277 (A and B) or with the isogenic nonfimbriated mutant JI-1 (C and D) (both strains used at 5 x 107 CFU). Peritoneal lavage was performed 20 h postinfection. Serial 10-fold dilutions of peritoneal fluid were plated onto blood agar plates with hemin/menadione and cultured anaerobically for enumerating recovered peritoneal CFU (A and C). The peritoneal fluid was also used to measure IL-12p70 and IFN-{gamma} production by ELISA (B and D). Data are shown for each mouse, and horizontal lines indicate the mean value (A) or are shown as means ± SD (n = 5) (B), from one of two experiments that yielded similar findings. Asterisks indicate significant (p < 0.05) differences in P. gingivalis peritoneal CFU (A) or in cytokine production (B).

 
Inhibition of P. gingivalis-induced periodontal bone loss by CR3 antagonist

Our in vitro and in vivo findings suggest that CR3 may be exploited by P. gingivalis in ways that promote its virulence. In the context of periodontal disease, the virulence of this pathogen is determined by its capacity to induce periodontal bone resorption in animal models (39). We thus used a validated model of mouse periodontitis (34) and hypothesized that the CR3 antagonist XVA143 suppresses P. gingivalis-induced periodontal bone loss. For this purpose, groups of five BALB/cByJ mice were microinjected into the palatal gingivae with XVA143 or PBS control and were orally infected with P. gingivalis or vehicle control, as detailed in Materials and Methods. In the groups microinjected with PBS, P. gingivalis infection resulted in significant periodontal bone loss compared with sham infection (p < 0.05; Fig. 10). However, the virulence of P. gingivalis in causing bone loss was significantly attenuated in mice microinjected with XVA143 (p < 0.05; Fig. 10). No significant differences were observed in sham-infected mice regardless of whether they were treated with XVA143 or PBS (Fig. 10). At termination of the experiment, viable P. gingivalis was recoverable by paper point sampling of the oral cavity of PBS-treated/infected, but not XVA143-treated/infected mice (viable cell counts = 3480 ± 1843 CFU, following anaerobic culturing on blood agar). This indicates that XVA143 inhibits the ability of P. gingivalis to persist in the mouse host. In conclusion, the CR3 antagonist XVA143 counteracts P. gingivalis virulence in the mouse periodontitis model.


Figure 10
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  		FIGURE 10. The CR3 antagonist, XVA143, prevents P. gingivalis-induced periodontal bone loss. BALB/cByJ mice were pretreated with XVA143 or PBS control and orally infected with P. gingivalis (P.g.) or 2% carboxymethylcellose vehicle only (Sham), as described in Materials and Methods. A, The mm distance from the CEJ to the ABC was measured at 14 predetermined sites in defleshed maxillae and was totaled for each mouse. Data are shown for each mouse, and horizontal lines indicate the mean value. B, The data from A were transformed to indicate bone loss, as outlined in Materials and Methods. Data are shown as means ± SD (n = 5), and negative values indicate bone loss. Asterisks denote significant (p < 0.05) differences between PBS-treated/P. gingivalis-infected mice and the rest of the groups, among which no significant differences were found.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our findings implicate CR3 in immune evasion by P. gingivalis. This pathogen exploits its fimbrial-mediated interaction with CR3 and proactively down-regulates IL-12 production via ERK1/2 signaling. The ability of P. gingivalis to escape immunosurveillance and promote its survival is counteracted by CR3 deficiency or a CR3 antagonist, or by lack of fimbria expression. The antagonist approach is of particular interest because it could be harnessed to negate immune evasion by P. gingivalis and thereby to attenuate its virulence in human periodontitis and possibly associated systemic conditions such as atherosclerosis. In a proof-of-concept study using the mouse periodontitis model, we showed that XVA143, an antagonist of both mouse and human CR3 (10, 31), inhibits the ability of P. gingivalis to persist in the mouse host and cause periodontal bone loss. Whether the attenuating effect of XVA143 on P. gingivalis virulence involves additional mechanisms cannot be formally ruled out. However, the fact that XVA143 is a potent CR3 antagonist that exerts similar effects on IL-12/IFN-{gamma} production and P. gingivalis clearance as CR3 deficiency does, strongly supports that the XVA143 effects are attributable, at least partly, to actions on CR3.

Suppression of IL-12p70 is most likely a major bacterial evasion strategy, because this cytokine induces the production of IFN-{gamma} and plays a key role in mediating bacterial clearance (16). The observation that P. gingivalis fimbria-exposed macrophages display diminished induction of IL-12p70 and IFN-{gamma} in response to other independent bacterial stimuli suggests that this evasion mechanism may impact on the survival of both P. gingivalis and other periodontal pathogens that cohabit its niche in mixed species biofilm. Fimbriae could act in cell-associated form, as free fimbriae shed from the cell surface, or as components of outer membrane vesicles released from the bacterial cell surface (40). Interestingly, an inverse relationship exists between IL-12p70 and severity of periodontal disease (41, 42). Moreover, removal of P. gingivalis by periodontal therapy results in selective augmentation of monocyte production of IL-12p70 (43), perhaps attributable in part to diminished P. gingivalis fimbria-CR3 interactions. It is thus possible that proactive inhibition of IL-12 induction may lead to P. gingivalis persistence, explaining, at least partly, the chronicity of periodontal disease. Although IL-23 did not seem to play a significant role in P. gingivalis clearance from systemically infected mice (Fig. 6C), this cytokine stimulates production of IL-17 by the Th17 T cell subset, and its involvement in periodontal host defense cannot be ruled out. In this context, a recent study using IL-17R knockout mice indicated an important role for innate responses and the Th17 axis of immunity in the control of P. gingivalis-induced bone loss (44).

Although CR3 exploitation by P. gingivalis appears to undermine effective innate immunity, it is unlikely to suppress inflammation altogether because the fimbria-CR3 interaction specifically down-regulates IL-12 (Fig. 3). In fact, inflammation could benefit P. gingivalis through acquisition of serum exudate-derived nutrients that are absolutely required for its growth (40). Inflammation that fails to clear the pathogen could be characterized as nonproductive and may underlie inflammatory periodontal bone resorption when infection becomes chronic.

From the host viewpoint, CR3-dependent inhibition of IL-12 appears to serve a physiological role. Indeed, the phagocytosis of apoptotic cells by macrophages is heavily dependent on CR3 and is associated with inhibition of IL-12p70, because apoptotic cells are not normally recognized as danger (45, 46). These findings in conjunction with our current data suggest that P. gingivalis has co-opted a natural anti-inflammatory CR3-dependent mechanism to evade innate immunity. This mechanism may be exploited also by other pathogens. For example, the interaction of B. pertussis filamentous hemagglutinin with CR3 similarly leads to inhibition of IL-12p70 (5), and, interestingly, the in vivo phagocytic uptake of B. pertussis via CR3 fails to promote its clearance (25).

P. gingivalis fimbriae and other microbial virulence proteins are not sensu stricto pathogen-associated molecular patterns, because they are responsible for adaptive fitness and thus do not represent invariant structures. This is in stark contrast to conserved molecular patterns that were selected by evolution as targets of pattern recognition (47). We and others have speculated that microbial virulence proteins have evolved to interact with and possibly exploit TLRs and the pattern recognition system in general, in ways that increase the potential of the pathogens for survival (8, 13, 14, 48, 49, 50, 51). In this regard, the interaction of P. gingivalis fimbriae with CR3 depends strictly upon the ability of fimbriae to exploit TLR2 inside-out signaling for activating the ligand-binding capacity of CR3 (9, 10) (Fig. 1A), and the pathogen colocalizes with both PRRs in macrophages (Fig. 1B). However, in addition to stimulating proadhesive inside-out signaling through Rac1/PI3K (9, 10), TLR2 also activates NF-{kappa}B through MyD88 (52). In fact, TLR2 deficiency abrogates IL-12p70, whereas CR3 deficiency augments IL-12p70 induction by P. gingivalis fimbriae (14), implying that CR3 engages in a cross-talk with TLR2 for IL-12 down-regulation. These findings suggest that under certain conditions TLR2 has the inherent potential for inducing protective immunity. Indeed, both in vitro and in vivo studies have shown that TLR2 mediates protection against various pathogens through induction of IL-12 and production of IL-12-dependent IFN-{gamma} (53, 54, 55, 56, 57). By contrast, TLR2 signaling has been implicated in immune evasion by Yersinia enterocolitica. Specifically, Y. enterocolitica expresses the virulence Ag LcrV, which induces immunosuppression through TLR2-dependent IL-10 release, resulting in enhanced pathogen survival (48). Moreover, a recent report has shown that TLR2 deficiency attenuates P. gingivalis-induced periodontal bone loss (58). The mechanism(s) whereby P. gingivalis exploits TLR2 to promote its virulence is largely uncertain at the moment. However, it is intriguing to suggest that TLR2 deficiency limits efficient activation of CR3, which in turn cannot be readily exploited by P. gingivalis in the way described in our study. This could explain, at least partly, why TLR2 deficiency attenuates P. gingivalis virulence in experimental periodontitis.

In our investigation of the role of CR3 in P. gingivalis-induced periodontal bone loss, we used a CR3 antagonist rather than a comparison of CR3-deficient mice with wild-type controls. The CR3 (CD11b–/–) deficiency is available on a C57BL/6 genetic background (The Jackson Laboratory), which is quite resistant to induction of periodontal bone loss, in contrast to the relatively susceptible BALB/c background (34). C57BL/6 mice are thus quite useful for examining receptor deficiencies that would predispose to susceptibility to periodontitis, rather than to resistance, which applied to our hypothesis with CR3. In the context of host receptor exploitation in periodontitis, therefore, BALB/c mice constitute a more useful model. There is an additional advantage in using the CR3 antagonist approach; if effective, as shown in our study, the antagonist can further be considered as a potential immunomodulatory agent for controlling human periodontitis. Interestingly, Baker et al. (59) used the relatively resistant C57BL/6 mice to determine the effect of adhesion molecule deficiencies on susceptibility to P. gingivalis-induced periodontal bone loss. Although deficiencies in P-selectin or ICAM-1 rendered the mutant mice susceptible to significant bone loss, a hypomorphic mutation in CD18 (resulting in diminished CR3 expression; 2–3% of wild-type levels (60)) did not predispose to bone loss. In fact, CD18-deficient mice displayed a trend for increased resistance to experimental periodontitis, although this could not be clearly shown in an already resistant genotype. Although CD18 deficiency affects more beta2 integrins (LFA-1 and p150,95) than just CR3, these data are consistent with the notion that CR3 is associated with increased P. gingivalis virulence in the periodontal bone loss model. It is moreover intriguing to suggest that the increased resistance of C57BL/6 mice to P. gingivalis-induced periodontitis, relative to BALB/c mice, could be attributable to the capacity of C57BL/6 mice to elicit higher levels of IL-12 and to exert enhanced bacterial clearance (61).

In conclusion, our findings indicate that CR3 is exploited by P. gingivalis for proactive suppression of the innate response in ways that promote its persistence and virulence. The ability of a CR3 antagonist (XVA143) to reverse suppression of IL-12p70, and thereby negate these immune evasion effects, holds promise for a potential therapeutic immunomodulation in human periodontitis and perhaps associated systemic diseases. Our results and their interpretation are in line with clinical research studies indicating an inverse relationship between IL-12p70 and severity of periodontitis, concluding that periodontal infection and resulting inflammation may perpetuate due to decreased IL-12p70 levels (41, 42, 62).


   Acknowledgment
 
We thank Pukar Ratti for excellent technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 U.S. Public Health Service Grants DE015254 and DE018292 (to G.H.) from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. George Hajishengallis, University of Louisville Health Sciences Center, 501 South Preston Street, Room 206, Louisville, KY 40292. E-mail address: g0haji01{at}louisville.edu Back

3 Abbreviations used in this paper: CR3, complement receptor 3; ABC, alveolar bone crest; CEJ, cementoenamel junction; CHO, Chinese hamster ovary; PRR, pattern-recognition receptor. Back

Received for publication May 17, 2007. Accepted for publication June 13, 2007.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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