Protease-Activated Receptor-2 Activation: A Major Role in the Pathogenesis of Porphyromonas gingivalis Infection

Abstract

We have investigated the specific contribution of protease-activated receptor-2 (PAR2) to host defense during Porphyromonas gingivalis infection. Culture supernatants from P. gingivalis strains 33277 and W50 provoked Ca2+ mobilization in cells transfected with PAR2 (PAR2-KNRK) and desensitized the subsequent responses to PAR2-selective agonist. In addition, culture supernatants of P. gingivalis E8 (RgpA/RgpB double knockout) did not cause calcium response in PAR2-KNRK cells, evidencing the involvement of the arginine-specific cysteine proteases RgpA and RgpB in PAR2 activation by P. gingivalis. Injection of P. gingivalis into mouse subcutaneous chambers provoked an increased proteolytic activity, which was inhibited by serine protease inhibitors. Fluids collected from chambers of P. gingivalis-injected mice were able to activate PAR2 and this activation was inhibited by serine protease inhibitors. P. gingivalis inoculation into subcutaneous chambers of wild-type mice induced an inflammatory response that was inhibited by a serine protease inhibitor and was significantly reduced in PAR2-deficient mice. Finally, mice orally challenged with P. gingivalis developed alveolar bone loss, which was significantly reduced in PAR2-deficient mice at 42 and 60 days after P. gingivalis infection. We conclude that PAR2 is activated on P. gingivalis infection, in which it plays an important role in the host inflammatory response. We have investigated the specific contribution of protease-activated receptor-2 (PAR2) to host defense during Porphyromonas gingivalis infection. Culture supernatants from P. gingivalis strains 33277 and W50 provoked Ca2+ mobilization in cells transfected with PAR2 (PAR2-KNRK) and desensitized the subsequent responses to PAR2-selective agonist. In addition, culture supernatants of P. gingivalis E8 (RgpA/RgpB double knockout) did not cause calcium response in PAR2-KNRK cells, evidencing the involvement of the arginine-specific cysteine proteases RgpA and RgpB in PAR2 activation by P. gingivalis. Injection of P. gingivalis into mouse subcutaneous chambers provoked an increased proteolytic activity, which was inhibited by serine protease inhibitors. Fluids collected from chambers of P. gingivalis-injected mice were able to activate PAR2 and this activation was inhibited by serine protease inhibitors. P. gingivalis inoculation into subcutaneous chambers of wild-type mice induced an inflammatory response that was inhibited by a serine protease inhibitor and was significantly reduced in PAR2-deficient mice. Finally, mice orally challenged with P. gingivalis developed alveolar bone loss, which was significantly reduced in PAR2-deficient mice at 42 and 60 days after P. gingivalis infection. We conclude that PAR2 is activated on P. gingivalis infection, in which it plays an important role in the host inflammatory response. The protease-activated receptors (PARs) belong to a family of G-protein-coupled, seven-transmembrane-domain receptors.1Nystedt S Emilsson K Wahlestedt C Sundelin J Molecular cloning of a potential proteinase activated receptor.Proc Natl Acad Sci USA. 1994; 91: 9208-9212Crossref PubMed Scopus (827) Google Scholar, 2Vu TK Hung DT Wheaton VI Coughlin SR Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2654) Google Scholar Activation of PARs occurs through proteolytic cleavage of the extracellular domain, resulting in generation of a new N-terminal tethered ligand.3Ossovskaya VS Bunnet NW Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (918) Google Scholar To date, four PARs have been identified: PAR1, PAR2, PAR3, and PAR4.4Coughlin SR Vu TK Hung DT Wheaton VI Characterization of a functional thrombin receptor: issues and opportunities.J Clin Invest. 1992; 89: 351-355Crossref PubMed Scopus (245) Google Scholar, 5Coughlin SR Thrombin signaling and protease-activated receptors.Nature. 2000; 407: 258-264Crossref PubMed Scopus (2110) Google Scholar For PAR1, PAR2, and PAR4, synthetic peptide agonists corresponding to the newly created N-terminus are able to activate the receptor in the absence of receptor cleavage.6Cocks TM Moffatt JD Proteinase-activated receptors: sentries for inflammation?.Trends Pharmacol Sci. 2000; 21: 103-108Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 7Corvera CU Dery O McConalogue K Bohm SK Khitin LM Caughey GH Payan DG Bunnett NW Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.J Clin Invest. 1997; 100: 1383-1393Crossref PubMed Scopus (267) Google Scholar Although these receptors have a similar mechanism of activation,8Coughlin SR Camerer E PARticipation in inflammation.J Clin Invest. 2003; 111: 25-27Crossref PubMed Scopus (218) Google Scholar they may have different biological functions and tissue distribution, and they can be activated by different proteases. PAR1, PAR3, and PAR4 are activated by thrombin and are implicated in platelet aggregation.8Coughlin SR Camerer E PARticipation in inflammation.J Clin Invest. 2003; 111: 25-27Crossref PubMed Scopus (218) Google Scholar Trypsin, mast cell tryptase, neutrophil protease 3, tissue factor/factor VIIa/factor Xa, membrane-tethered serine protease-1, and gingipains have been identified as activators of PAR2.9Lourbakos A Potemba J Travis J D'Andrea MR Andrade-Gordon P Santulli R Mackie EJ Pike RN Arginine-specific proteinase from Porphyromonas gingivalis activates proteinase-activated receptors on human oral epithelial cells and induces interlukin-6 secretion.Infect Immun. 2001; 69: 5121-5130Crossref PubMed Scopus (196) Google Scholar, 10Vergnolle N Modulation of visceral pain and inflammation by protease-activated receptors.Br J Pharmacol. 2004; 141: 1264-1274Crossref PubMed Scopus (81) Google Scholar, 11Uehara A Muramoto K Takada H Sugawara S Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through proteinase-activated receptor 2.J Immunol. 2003; 170: 5690-5696PubMed Google Scholar Some studies have shown that PAR2 participates in inflammatory processes in vivo.12Vergnolle N Wallace JL Bunnett NW Hollemberg MD Proteinase-activated receptors in inflammation, neuronal signaling and pain.Trends Pharmacol Sci. 2001; 22: 146-152Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 13Vergnolle N Hollenberg MD Sharkey KA Wallace JL Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR-2)-activating peptides in the rat paw.Br J Pharmacol. 1999; 127: 1083-1090Crossref PubMed Scopus (202) Google Scholar Although PAR2 activation causes edema and granulocyte recruitment in the rat paw,13Vergnolle N Hollenberg MD Sharkey KA Wallace JL Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR-2)-activating peptides in the rat paw.Br J Pharmacol. 1999; 127: 1083-1090Crossref PubMed Scopus (202) Google Scholar the role of PAR2 on mucosal surfaces is not as clearly defined. PAR2 agonists exert protective effects in airways against lipopolysaccharide challenge14Cocks TM Fong B Chow JM Anderson GP Frauman AG Goldie RG Henry PJ Carr MJ Hamilton JR Moffatt JD A protective role for protease-activated receptors in the airways.Nature. 1999; 398: 156-160Crossref PubMed Scopus (324) Google Scholar and in the colon in a model of chronic colitis induced by trinitrobenzene sulfonic acid,15Fiorucci S Mencarelli A Palazzetti B Distrutti E Vergnolle N Hollenberg MD Wallace JL Morelli A Cirino G Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis.Proc Natl Acad Sci USA. 2001; 98: 13936-13941Crossref PubMed Scopus (182) Google Scholar but they are proinflammatory when acutely administered in the colon16Cenac N Coelho AM Nguyen C Compton S Andrade-Gordon P MacNaughton WK Wallace JL Hollenberg MD Bunnett NW Garcia-Villar R Bueno L Vergnolle N Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2.Am J Pathol. 2002; 161: 1903-1915Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar or in the airways17Schmidlin F Amadesi S Dabbagh K Lewis DE Knott P Bunnett NW Gater PR Geppetti P Bertrand C Stevens ME Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway.J Immunol. 2002; 169: 5315-5321PubMed Google Scholar of mice. Recently, some studies have suggested a role for PAR2 in periodontitis, a chronic oral inflammation leading to bone and tooth loss, because it was found to be expressed by osteoblasts, oral epithelial cells, and human gingival fibroblasts.9Lourbakos A Potemba J Travis J D'Andrea MR Andrade-Gordon P Santulli R Mackie EJ Pike RN Arginine-specific proteinase from Porphyromonas gingivalis activates proteinase-activated receptors on human oral epithelial cells and induces interlukin-6 secretion.Infect Immun. 2001; 69: 5121-5130Crossref PubMed Scopus (196) Google Scholar, 11Uehara A Muramoto K Takada H Sugawara S Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through proteinase-activated receptor 2.J Immunol. 2003; 170: 5690-5696PubMed Google Scholar, 18Abraham LA Chinni C Jenkins AL Lourbakos A Ally N Pike RN Mackie EJ Expression of protease-activated receptor-2 by osteoblasts.Bone. 2000; 26: 7-14Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Importantly, Lourbakos and colleagues9Lourbakos A Potemba J Travis J D'Andrea MR Andrade-Gordon P Santulli R Mackie EJ Pike RN Arginine-specific proteinase from Porphyromonas gingivalis activates proteinase-activated receptors on human oral epithelial cells and induces interlukin-6 secretion.Infect Immun. 2001; 69: 5121-5130Crossref PubMed Scopus (196) Google Scholar reported that bacterial cysteine proteases such as Arg-gingipain (Rgp), which are produced by Porphyromonas gingivalis (a major causative agent of chronic periodontitis), was able to activate PAR2 in oral epithelial cells and to induce the secretion of the proinflammatory cytokine interleukin (IL)-6, which is a potent stimulator of osteoclast differentiation and bone resorption. The production of potent proinflammatory mediators was also shown by Uehara and colleagues11Uehara A Muramoto K Takada H Sugawara S Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through proteinase-activated receptor 2.J Immunol. 2003; 170: 5690-5696PubMed Google Scholar who demonstrated that a synthetic PAR2 agonist peptide activated the production of IL-8 in human gingival fibroblasts. In a previous study,19Holzhausen M Spolidorio LC Vergnolle N Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats.J Dent Res. 2005; 84: 154-159Crossref PubMed Scopus (41) Google Scholar we have evaluated the in vivo effects of PAR2 activation by a selective agonist (SLIGRL-NH2) on periodontal disease in rats. The PAR2 agonist caused periodontitis (inflammation and alveolar bone loss) through a mechanism involving prostaglandin release and matrix metalloproteinase activation. Taken together, these studies strongly suggest a role for PAR2 activation in inducing inflammation and bone resorption during periodontitis. However, a study by Smith and colleagues20Smith R Ransjo M Tatarczuch L Song SJ Pagel C Morrison JR Pike RN Mackie EJ Activation of protease-activated receptor-2 leads to inhibition of osteoclast differentiation.J Bone Miner Res. 2004; 19: 507-516Crossref PubMed Scopus (32) Google Scholar in 2004 suggested an opposite role for PAR2 activation: they hypothesized that PAR2 activation may inhibit bone resorption in the context of periodontal diseases. They showed that PAR2 agonists inhibited osteoclast differentiation, therefore being possibly protective against bone loss signals. Interestingly, Chung and colleagues21Chung WO Hansen SR Rao D Dale BA Protease-activated receptor signaling increases epithelial antimicrobial peptide expression.J Immunol. 2004; 173: 5165-5170Crossref PubMed Scopus (90) Google Scholar have identified PAR2 as a receptor used by Rgp in induction of human β-defensin-2 (hBD-2) in oral epithelial cells and have also demonstrated hBD-2 induction by PAR2 peptide agonist. Notably, antimicrobial peptides of the hBD family are part of the innate immune responses that play a role in mucosal defense. No definitive answer on the role of PAR2 in oral mucosal inflammation or in periodontal diseases (pro- or anti-inflammatory effect, pro- or anti-bone loss signal) has so far emerged. Therefore, our strategy was to use a genetic approach, using PAR2-deficient mice, to study the specific contribution of PAR2 activation and proteolytic activity during infection by the proteolytic periodontal pathogen P. gingivalis. PAR2-deficient (PAR2−/−) and wild-type (WT) littermate (PAR2+/+) mice, both of the C57BL6 background22Damiano BP Cheung WM Santulli RJ Fung-Leung WP Ngo K Ye RD Darrow AL Derian CK de Garavilla L Andrade-Gordon P Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1.J Pharmacol Exp Ther. 1999; 288: 671-678PubMed Google Scholar originally obtained from Johnson and Johnson Pharmaceutical Research Institute (Spring House, PA), were bred at the University of Calgary animal care facility. Six- to nine-week-old mice were used in the present study. All animals were housed in a temperature-controlled room; food and water were provided ad libitum. The Animal Care and Ethics Committees of the University of Calgary approved all experimental protocols, which followed the guidelines of the Canadian Council on Animal Care. P. gingivalis strains 33277 [American Type Culture Collection (ATCC), Rockville, MD], W50 (WT), and E8 (RgpA/RgpB double mutant, obtained from Dr. J. Aduse-Opoku, Queen Mary's School of Medicine and Dentistry, London, UK) were grown on anaerobic blood agar plates in an anaerobic chamber with 85% N2, 5% H2, and 10% CO2. After incubation at 37°C for 7 days, the bacteria were collected and suspended in Schaedler broth (Difco Laboratories, Detroit, MI) to a final optical density of 1.2 (109 CFU/ml) at 660 nm. Treponema denticola strain ATCC 35405 was obtained from the culture collection at the University of Toronto. It was grown in modified new oral spirochete medium23Dawson JR Ellen RP Tip-oriented adherence of Treponema denticola to fibronectin.Infect Immun. 1990; 58: 3924-3928Crossref PubMed Google Scholar for 3 days (mid-exponential phase) to a concentration of 2.8 × 109 cells/ml, as determined by microscopic count. Coil-shaped chambers were prepared from 0.5-mm stainless-steel wire and were surgically implanted in the subcutaneous tissue of the dorso-lumbar region of each mouse, as previously described.24Gyurko R Boustany G Huang PL Kantarci A Van Dyke TE Genco CA Gibson FC Mice lacking inducible nitric oxide synthase demonstrate impaired killing of Porphyromonas gingivalis.Infect Immun. 2003; 71: 4917-4924Crossref PubMed Scopus (42) Google Scholar Ten days after implantation, chambers were inoculated with 0.1 ml of P. gingivalis 33277, P. gingivalis W50, or P. gingivalis E8, suspended in sterile saline (109 cells/ml). Control mice were inoculated with vehicle only. Mice were sacrificed by cervical dislocation at 1 day after bacterial inoculation. Another group of mice was inoculated with 0.1 ml of a solution containing P. gingivalis (109 cells/ml) and 0.3 mg/ml of SBTI (soybean trypsin inhibitor; Sigma, St. Louis, MO) and sacrificed 1 day after. A sample of chamber fluid (100 μl) was aseptically collected from each animal at 1 day after P. gingivalis or vehicle challenge to assess the host inflammatory response. A 10-μl aliquot of each chamber fluid was diluted 10-fold in Turk's staining solution to determine the total number of inflammatory cells by light microscopy, using a Neubauer counting chamber (Hauser Scientific, Horsham, PA). The rest of the samples were aliquoted and kept for evaluation of proteolytic activity as well as cytokine and prostaglandin release. PAR2-expressing Kirsten sarcoma-transformed rat kidney epithelial cells (KNRK: ATCC, Manassas, VA) were propagated in geneticin (0.6 mg/ml)-containing medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% Penstrep) to maintain vector-selective pressure. Nontransfected KNRK cells were grown in geneticin-free medium. Cells at 90% confluence in 80-cm2 flasks (Life Technologies, Inc., Grand Island, NY) were rinsed with phosphate-buffered saline, lifted with nonenzymatic cell dissociation fluid, and pelleted before resuspension in 1 ml of Hanks' balanced salt solution (pH 7.4), 2.5 μl of sulfinpyrazone (100 mmol/L), 1 μl of a 20% Pluronic F-127 solution, and 10 μl of 2.5 mg/ml Fluo-3 acetoxymethylester (Molecular Probes, Inc., Eugene, OR). The final solution was incubated at room temperature while shaking gently for 25 minutes. Cells were then washed three times and resuspended in calcium assay buffer (150 mmol/L NaCl, 3 mmol/L KCl, 1.5 mmol/L CaCl2, 10 mmol/L glucose, 20 mmol/L HEPES, 0.25 sulfinpyrazone, pH 7.4). Determination of intracellular calcium mobilization was performed as previously described.25Compton SJ Cairns JA Palmer KJ Al-Ani B Hollenberg MD Walls AF A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists.J Biol Chem. 2000; 275: 39207-39312Crossref PubMed Scopus (69) Google Scholar Briefly, fluorescence measurements were performed on a fluorescence spectrometer 650-10S (Perkin-Elmer, Norwalk, CT). Suspensions of cells (5 × 105 cells/ml) loaded with the calcium fluorochrome fluo-3 (Molecular Probes, Inc.) were placed in 4-ml cuvettes, stirred with a magnetic flea bar, and maintained at 24°C. The signal produced by PAR2-expressing KNRK or nontransfected KNRK cells was measured after the addition of culture supernatants (40 μl) of P. gingivalis 33277, W50, or E8, and T. denticola 35405. In addition, we also evaluated the signal produced by PAR2-expressing KNRK with the addition of supernatants from chamber fluid samples, previously incubated with either 10 nmol/L SBTI (trypsin inhibitor), 1 mmol/L PMSF (phenylmethyl sulfonyl fluoride) (serine proteinase inhibitor, Sigma), 1 mmol/L TLCK (N-p-tosyl-l-lysine chloromethyl ketone, cysteine proteinase inhibitor; Sigma), or 1 mmol/L leupeptin (Rgp cysteine proteinase inhibitor, Sigma) for 10 minutes. The calcium mobilization after the addition of culture supernatants of P. gingivalis 33277, W50, or E8 preincubated with either 1 mmol/L TLCK or 1 mmol/L leupeptin for 10 minutes was evaluated. The calcium responses to known PAR2 agonists [2 nmol/L trypsin (Sigma) or 2 μmol/L of SLIGRL-NH2] were also evaluated. The results are expressed as percentages (% A23187) of the fluorescence emission (E530) caused by 2-μmol/L concentrations of the calcium ionophore A23187. The PAR2 agonist peptide was synthesized by the Peptide Synthesis Facility (University of Calgary, Calgary, AB, Canada). All experiments were repeated three to five times, for at least eight samples. After centrifugation of the samples from the fluid chambers, supernatants were collected and assayed for proteolytic activity using a Fluoroskan Ascent fluorimeter (Thermo Electron, Franklin, MA). Besides testing the proteolytic activity of the supernatants of chamber fluid samples, we also evaluated the effect of preincubation (10 minutes) of the samples with either 10 nmol/L SBTI (trypsin inhibitor), 1 mmol/L PMSF (serine proteinase inhibitor, Sigma), 1 mmol/L TLCK (serine and cysteine proteinase inhibitor, Sigma), or 1 mmol/L leupeptin (serine and Rgp cysteine proteinase inhibitor, Sigma). Assays were performed at 25°C, with a 355-nm excitation wavelength filter and a 460-nm emission wavelength filter. Fluorescence from wells on the microplate was measured throughout 20 minutes. The hydrolysis of the substrate (5 μl) added to sample solution (75 μmol/L Boc-Gln-Ala-Arg-AMC in 50 mmol/L Tris/20 mmol/L CaCl2 buffer, pH 7.4) was calibrated with the rate of AMC hydrolysis product in a standard reaction mixture using a serial dilution of trypsin (0.05 to 2 U/ml). All assays were performed in triplicate; the range of values observed was always less than 10% of the mean. The results are expressed in units of trypsin per milliliter (U/ml). Prostaglandin-E2, IFN-γ, IL-1, IL-6, and IL-10 levels in the supernatants of chamber fluid samples collected from mice that received inoculation of P. gingivalis 33277 or vehicle were determined by using commercially available enzyme-linked immunosorbent assay kits according to manufacturer's instructions (R&D Systems, Minneapolis, MN). The concentration of the inflammatory mediators was determined using the Softmax data analysis program (Molecular Devices, Menlo Park, CA). A total of 0.2 ml of P. gingivalis 33277 suspended in sterile saline (109 cells) was given to each mouse via a gavage needle every other day for a total of 4 days, in part by gavage, and by local application in the oral cavity. Mice were then allowed free access to standard mouse chow and water. The mice were sacrificed by cervical dislocation 42 and 60 days after the last bacterial administration. Ten sham-infected (sterile saline-injected) and 10 P. gingivalis-infected mice were used at each time point in each animal group (PAR2−/− or WT mice). Mandibles were removed, hemisected, exposed to NaOH (2N), and then mechanically defleshed. Horizontal bone loss around the mandibullary molars was assessed by measuring the distance between the cementoenamel junction and alveolar bone crest under a dissecting microscope (×40). Measurements of bone level were done at seven sites on the lingual side of the left and right mandibula molars, and a total of 14 measurements per mouse were done three times in a random, blinded protocol by one evaluator. One-way analysis of variance was used to compare means among groups. In case of significant differences among the groups, posthoc two-group comparisons were assessed with Tukey-Kramer test. A P value <0.05 was considered statistically significant. Data are expressed as mean ± SE. To evaluate the role of proteolytic activity produced by the bacteria itself, we examined the effects of P. gingivalis 33277, P. gingivalis W50, and P. gingivalis E8 culture supernatants on Ca2+ mobilization in PAR2-KNRK cells (Figure 1). We first confirmed that in the KNRK cell line, no calcium mobilization was observed after addition of any of the P. gingivalis culture supernatants or trypsin (data not shown). Then, we showed that P. gingivalis culture supernatants (40 μl) from strains 33277 and W50 provoked Ca2+ mobilization in PAR2-KNRK cells. However, the calcium response induced by culture supernatants of P. gingivalis (33277 and W50) was significantly lower than the response to 2 μmol/L of the selective agonist SLIGRL-NH2 (Figure 1A). Preincubation of PAR2-KNRK cells with culture supernatants of P. gingivalis (33277 and W50) significantly reduced the subsequent response of those cells to SLIGRL-NH2 (Figure 1A). We demonstrated that after desensitization of PAR2 receptor by two sequential treatments with trypsin for 5 minutes, the addition of P. gingivalis 33277 culture supernatant did not cause any calcium response in PAR2-KNRK cells (Figure 1B, XI). Taken together, these results show that proteases released by P. gingivalis 33277 and P. gingivalis W50 activate PAR2 in PAR2-transfected cells. The involvement of the cysteine proteases Arg-gingipain in PAR2 activation was delineated by two different approaches. First we used the culture supernatants from strains P. gingivalis W50 (WT) and E8 (RgpA/RgpB double knockout). We found that P. gingivalis W50 culture supernatants (40 μl) provoked Ca2+ mobilization in PAR2-KNRK cells and decreased the subsequent responses to SLIGRL-NH2 or trypsin (Figure 1B, III and VIII). No significant difference was found between the two strains of P. gingivalis (33277 and W50, Figure 1A). However, P. gingivalis E8 culture supernatant (40 μl) did not cause calcium response in PAR2-KNRK cells and did not alter the subsequent responses to trypsin or SLIGRL-NH2 (Figure 1B, IV and IX; not shown for SLIGRL-NH2). Then, we found that preincubation of P. gingivalis 33277 and P. gingivalis W50 culture supernatant with two serine/cysteine protease inhibitors, TLCK and leupeptin, led to a significant increase in the subsequent response to SLIGRL-NH2 compared to untreated supernatants (Figure 1A). The P. gingivalis 33277 and P. gingivalis W50 culture supernatant-induced PAR2 desensitization was inhibited by serine/cysteine protease inhibitors (leupeptin and TLCK). Taken together, these results implicate the arginine-specific cysteine proteases RgpA and RgpB in PAR2 activation. The ability to activate PAR2 exerted by P. gingivalis was compared with the effects exerted by T. denticola, a periodontal pathogen that expresses a serine protease in its outer sheath and several endo-acting peptidases in its culture supernatants. The T. denticola culture supernatant did not cause calcium signal in PAR2-transfected cells (Figure 1B, V). However, the trypsin response of PAR2-transfected cells pre-exposed to T. denticola culture supernatant was diminished (Figure 1B, X), whereas the response to the peptide agonist SLIGRL-NH2 was similar to that of cells only exposed to the peptide (Figure 1B, V). This suggests that proteases released in the supernatant of T. denticola cultures do not activate PAR2 but may disarm the receptor for subsequent activation by trypsin. We hypothesized that PAR2 can be activated upon P. gingivalis infection, thereby participating in the host response to infection. To this end, we first evaluated whether P. gingivalis infection provoked the release of proteolytic activity. Trypsin-like activity was tested in fluids collected from skin chambers after inoculation of P. gingivalis 33277, P. gingivalis W50, P. gingivalis E8, or saline. In Figure 2, WT mice showed a significant (P < 0.001) increase with regards to the proteolytic activity of chamber fluids after inoculation with P. gingivalis 33277, P. gingivalis W50, or P. gingivalis E8 when compared to chamber fluid from mice injected with saline (control). Yet, the proteolytic activity from samples of P. gingivalis E8-treated mice was significantly decreased when compared to the proteolytic activity found in chamber fluids after inoculation with P. gingivalis 33277 or P. gingivalis W50. Preincubation with the serine protease inhibitors SBTI or PMSF led to a significant reduction of the proteolytic activity on P. gingivalis 33277, P. gingivalis W50, or P. gingivalis E8 infection, whereas preincubation with TLCK or leupeptin showed a significant decrease only in the proteolytic activity of samples from P. gingivalis 33277-infected mice. In addition, systemic treatment of mice with the serine protease inhibitor SBTI significantly (P < 0.05) decreased the proteolytic activity because of P. gingivalis 33277 inoculation (mean ± SEM, 0.44 ± 0.23 versus 1.56 ± 0.21 for P. gingivalis 33277 + SBTI and P. gingivalis 33277 alone, respectively, data not shown in the figure). To determine whether or not proteolytic activity released on P. gingivalis infection could cleave PAR2, we examined the effects of P. gingivalis-infected chamber fluid samples on subsequent intracellular calcium [Ca2+]i mobilization by SLIGRL-NH2 in cultured cells transfected with PAR2 (PAR2-KNRK). We observed that chamber fluid samples (40 μl) collected from WT mice after subcutaneous challenge with P. gingivalis 33277 or P. gingivalis W50 provoked calcium mobilization in PAR2-KNRK but not in nontransfected (KNRK) cells (data not shown). Fluids collected from P. gingivalis 33277- or P. gingivalis W50-infected mice but not from P. gingivalis E8-injected mice were able to significantly (P < 0.05) reduce calcium mobilization in PAR2-transfected KNRK cells generated by SLIGRL-NH2 (mean ± SEM, 12.38 ± 1.35, 12.35 ± 1.55, 22.10 ± 2.12 for P. gingivalis 33277, P. gingivalis W50, and P. gingivalis E8 treatment, respectively; Figure 3), as compared to the effects of saline-injected chambers (mean ± SEM, 29.13 ± 0.30). Preincubation of the P. gingivalis 33277-infected or P. gingivalis W50-infected chamber samples with SBTI or PMSF significantly increased the subsequent calcium response to SLIGRL-NH2 (Figure 3), thereby inhibiting the desensitization to SLIGRL-NH2 response. Preincubation of chamber fluid samples with TLCK or leupeptin also provoked a significant increase in the subsequent calcium response to SLIGRL-NH2, but only in samples from P. gingivalis 33277-infected mice. Fluid samples collected from mice treated with both P. gingivalis 33277 and SBTI did not provoke a significant reduction in the subsequent calcium signal response to SLIGRL-NH2 (mean ± SEM, 21.56 ± 1.72; data not shown), similar to the samples from P. gingivalis E8-treated and control-treated animals. Collectively, these results demonstrated the presence of PAR2-activating proteases upon infection by P. gingivalis. Next, we evaluated whether PAR2 activation affected the host response to P. gingivalis infection, by comparing the inflammatory effects of subcutaneous chamber infection with P. gingivalis 33277 in WT and PAR2−/− mice. Figure 4A shows that subcutaneous challenge with P. gingivalis led to a significant (P < 0.001) increase in inflammatory cell infiltration when compared to saline treatment (day 1 after saline or P. gingivalis injection) in WT mice. P. gingivalis-induced increased inflammatory cell counts were significantly diminished in PAR2−/− mice comp