Kinin Danger Signals Proteolytically Released by Gingipain Induce Fimbriae-Specific IFN-γ- and IL-17-Producing T Cells in Mice Infected This information is current as Intramucosally with Porphyromonas of September 26, 2021. gingivalis Ana Carolina Monteiro, Aline Scovino, Susane Raposo, Vinicius Mussa Gaze, Catia Cruz, Erik Svensjö, Marcelo

Sampaio Narciso, Ana Paula Colombo, João B. Pesquero, Downloaded from Eduardo Feres-Filho, Ky-Anh Nguyen, Aneta Sroka, Jan Potempa and Julio Scharfstein J Immunol 2009; 183:3700-3711; Prepublished online 17 August 2009;

doi: 10.4049/jimmunol.0900895 http://www.jimmunol.org/ http://www.jimmunol.org/content/183/6/3700

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Kinin Danger Signals Proteolytically Released by Gingipain Induce Fimbriae-Specific IFN-␥- and IL-17-Producing T Cells in Mice Infected Intramucosally with Porphyromonas gingivalis1

Ana Carolina Monteiro,* Aline Scovino,* Susane Raposo,*† Vinicius Mussa Gaze,*† Catia Cruz,* Erik Svensjo¨,* Marcelo Sampaio Narciso,‡ Ana Paula Colombo,§ Joa˜o B. Pesquero,¶ Eduardo Feres-Filho,† Ky-Anh Nguyen,ʈ# Aneta Sroka,** Jan Potempa,**†† and Julio Scharfstein2*

Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine pro- tease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, we Downloaded from studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B2 receptors (B2R). Our data show that P. gingivalis W83 (wild type), but not gingipain-deficient mutant or wild-type bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in BALB/c or C57BL/6 mice. ؊/؊ ؊/؊ Studies in TLR2 ,B2R , and neutrophil-depleted C57BL/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a readout to verify whether activation http://www.jimmunol.org/ 3 3 of the TLR2 PMN B2R axis (where PMN is polymorphonuclear neutrophil) at early stages of mucosal infection had impact ␥ on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B2R-dependent generation of IFN- - producing Fim T cells in submandibular draining lymph nodes of BALB/c and C57BL/6 mice, whereas IL-17-producing Fim T cells were generated only in BALB/c mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forge a trans-cellular cross-talk between TLR2 and B2R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify whether kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis. The Journal of Immunology, 2009, 183: 3700–3711. by guest on September 26, 2021 eriodontal disease is initiated by a chronic subgingival gingivalis, whereas the normal flora is dominated by facultative bacterial infection that destroys the underlying alveolar Gram-positive bacteria (1). Studies in laboratory animals infected P bone as well as the connective tissue that attaches the gin- with P. gingivalis (1) suggest that bacteria are required for the giva to the tooth root (1). During the progression of periodontitis, initiation of inflammation, whereas bone erosion is more likely composition of the subgingival biofilm is changed; there is an en- caused by aberrant immune responses to bacterial Ags persistent in richment of anaerobic Gram-negative bacteria belonging to the red the periodontal tissues. The latter hypothesis received indirect sup- complex group of periodontal pathogens, such as Porphyromonas port from pathogenesis studies in animal models of autoimmune arthritis, showing that activated T and B cells up-regulate RANKL (receptor activator of NF-␬B ligand), thus being capable of driving *Carlos Chagas Filho Institute of Biophysics, †Faculty of Odontology, ‡Department the differentiation of bone-erosive osteoclasts (2, 3). Efforts to of Histology, Institute of Biomedical Sciences, and §Pablo de Go´es Institute of Mi- crobiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; ¶Department characterize osteoclastogenic T cell subsets have recently con- ʈ of Biophysics, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil; Institute of Dental verged at TH17 (4), a proinflammatory helper T cell subset whose Research, Westmead Millenium Institute and #Faculty of Dentistry, University of differentiation depends on the cooperative action of IL-6, TGF-␤, Sydney, Sydney, Australia; **Faculty of Biochemistry, Biophysics, and Biotechnol- ogy, Department of Microbiology, Jagiellonian University, Krakow, Poland; and and IL-23 (5–9). Although the severity of chronic human peri- ††Department of Periodontics, University of Louisville School of Dentistry, Louis- odontal disease is positively correlated with IL-17 levels (10–12), ville, KY 40202 the hypothesis that osteoclastogenic human TH17 cells have a Received for publication March 20, 2009. Accepted for publication July 8, 2009. causal role in alveolar bone erosion remains to be confirmed. The costs of publication of this article were defrayed in part by the payment of page As a recently recognized a risk factor in the development of charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. atherosclerosis, both in humans and in animal models (13, 14), P. gingivalis relies on multiple virulence factors, such as fimbriae 1 This work was supported by grants from Instituto Nacional de Pesquisa em Biologia 3 Estrutural e Bio-Imagem do Conselho Nacional de Desenvolvimento Científico e (Fim), LPS, cysteine , and hemagglutinin, to colonize Tecnolo´gico), Fundac¸a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro/Programa Pensa Rio, and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nível Superior, National Institutes of Health Grants DE 09761 and 1642/B/P01/2008/35, and a 3 Abbreviations used in this paper: Fim, fimbriae; BK, bradykinin; B2R, BK B2 re- grant from the Department of Scientific Research, Polish Ministry of Science and ceptor; DC, dendritic cell; HCP, hamster cheek pouch; HK, human kininogen; LBK, Education. lysyl-BK; LN, lymph node; NOD, nucleotide-binding oligomerization domain; p.i., 2 Address correspondence and reprint requests to Dr. Julio Scharfstein, Universidade postinfection; PMN, polymorphonuclear neutrophil; Rgps, arginine-specific gingi- Federal do Rio de Janeiro, Centro de Ciencias de Saude, Ilha do Fundao, Bloco D, pain; TAFI, thrombin-activatable fibrinolysis inhibitor; WT, wild type. Sala D 007, Rio de Janeiro, Rio de Janeiro 21944-900, Brazil. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900895 The Journal of Immunology 3701 and survive in oral mucosal tissues (15). Molecular studies led to Analysis of host resistance mechanisms in mice acutely infected the characterization of two major TLR2 ligands of P. gingivalis:an with T. cruzi (i.p. route) demonstrated that splenic DCs activated atypical LPS (16) and Fim, a multiunit complex composed of po- via the kinin/B2R pathway play a critical role in the generation of lymerized fimbrillin (FimA) and accessory proteins (FimCDE) immunoprotective IFN-␥-producing T cells (47). (17). More recently, analysis of macrophage responses to P. gin- For this report, we studied the impact of kinin system activation givalis or to its purified wall components (Fim and LPS) revealed on T cell development in mice challenged by a low intrabuccal that NF␬B-responsive genes were activated via TLR2- or TLR7- dose of P. gingivalis. Analysis of the dynamics of the inflamma- MyD88-p38 MAPK pathways, respectively (18). In another in tory response evoked by this periodontal pathogen revealed that vitro study, it was shown that Fim provide adhesive filamentous plasma leakage, a vascular response orchestrated by TLR2/neutro- appendages for gingival epithelial cells without triggering TLR2- phil activation at very early stages of infection, is a limiting step mediated proinflammatory responses, because these particular host governing the extravascular levels of immunostimulatory kinins cell types do not express the CD14 required for TLR2 released by gingipain. Our study provides the first demonstration signaling (19). Furthermore, recent studies showed that P. gingi- that activation of the kinin proteolytic network by a periodontal valis relies on native Fim to persist in mice infected i.p., the pathogen may shape the decision-making process underlying ef- immune subversion mechanism being ascribed to the TLR2/PI3K- fector T cell commitment in intramucosally infected mice. dependent up-regulation of CR3, a surface receptor that down- modulates intracellular killing by macrophages (20, 21). Materials and Methods Cysteine proteases from the gingipain family are responsible for All animal experiments were in accordance with National Institutes of

Ͼ85% of the general proteolytic activity generated by P. gingivalis Health Guidelines for animal use in research. Downloaded from (22). Biochemical studies (23) characterize arginine-specific gin- Mice and bacterial strains gipains (Rgps; RgpA and RgpB) of 95 and 50 kDa, both of which cleave Arg-Xaa peptide bonds (24), and a lysine-specific gingipain Experiments were done with mouse strains BALB/c, C57BL/6 wild type (WT; B Rϩ/ϩ and TLR2ϩ/ϩ), C57BL/6 B RϪ/Ϫ, and C57BL/6 TLR2Ϫ/Ϫ. (Kgp) of 105 kDa that specifically hydrolyzes Lys-Xaa peptide 2 2 The P. gingivalis W83 strain and its Rgps-deficient mutant (strain ⌬ Rgps; bonds (25). Gingipains promote bacterial virulence through a wide obtained by isogenic deletion of rgpA and rgpB genes) were grown anaer- range of molecular mechanisms, such as disruption of plasma clot- obically at 37°C in brain heart infusion broth with 0.5% (w/v) yeast extract http://www.jimmunol.org/ ting (26–28), alterations of complement system function (29, 30), and supplemented with cysteine (0.5 g/L), hemin (5 mg/L), and menadione modification of neutrophil functions (31) conversion of profimbri- (1 mg/L) (Sigma-Aldrich). The ⌬ Rgps mutant was created by truncating RgpB in a rgpA-deficient strain of P. gingivalis W83 by deletional mu- lin to mature fimbrillin (32), and degradation of antibacterial pep- tagenesis as described previously (50). Briefly, a primer set (5Ј-ATGA tides and host cell surface receptors (33–36). GAGTATCGCTGATTAATTCACACTGCAATTCTCTAATAAGG-3Ј, More recently, studies with human monocytic cell lines revealed 5Ј-TAATTCACACTGCAATTCTCTAATAAGGGC-3Ј,5Ј-CAGCGA that gingipains, acting synergistically with microbial-borne ligands TACTCTCATTTAATTTGATGATAGCCTTACCG-3Ј, and 5Ј-TAAT Ј of TLR and nucleotide-binding oligomerization domain (NOD) 2 TTGATGATAGCCTTACCGTCTTTCACG-3 ) was used to truncate RgpB at residue 410 in the master pURgpB-E plasmid by the site- receptors, may stimulate innate immunity through the induction of directed, -independent mutagenesis (SLIM) method of mutagen- proinflammatory chemokines via signaling of -activated esis (50). Homologous recombination of the modified plasmid into the by guest on September 26, 2021 receptors (37). The dichotomous effects of gingipains on host- rgpA-deficient strain resulted in the total loss of RgpB as determined by pathogen balance are further highlighted by evidence that these enzymatic assay and Western blot analysis. cysteine proteases increase vascular permeability through the ac- Bacterial molecules tivation of the kinin system (38, 39). Kinins are nonapeptide/de- capeptide hormones liberated proteolytically from an internal seg- Protein-free LPS from P. gingivalis W83 was extracted with phenol-water and purified by cesium chloride density gradient ultracentrifugation fol- ment of high or low m.w. kininogens. Whether released by plasma, lowed by repurification (51). The purified P. gingivalis LPS preparation tissue kallikreins, or microbial cysteine proteases such as gingi- was free of contamination by proteins, DNA, and RNA, which were de- pains, kinins exert their homeostatic or proinflammatory activities graded during extensive incubation of the LPS-containing fraction with through the signaling of the bradykinin (BK) B receptor (B R) proteinase K, DNase and RNase, respectively. The final preparation of LPS 2 2 stimulated the transcription of IL-1␤ and TNF-␣ in dose-dependent manner and B1 receptor, i.e., pharmacologically distinct subtypes of a G in human monocyte-derived macrophages, and this effect was abrogated by protein-coupled kinin receptor that are respectively triggered by polymyxin B, thus excluding the presence of TLR ligands other than LPS BK/lysyl-BK (LBK) and by des-Arg-BK/LBK, a processed me- in our sample. Fim from P. gingivalis 332277 were purified according to a tabolite generated by M/N (40, 41). Although method described previously (52). Briefly, P. gingivalis strains harvested B R is constitutively expressed by a broad range of cells, including from early stationary cultures by centrifugation were subjected to ultra- 2 sonication in an ice bath. Following centrifugation, Fim were purified from endothelial cells, smooth muscle cells, pain-sensitive neurons (42), the supernatant by ammonium sulfate precipitation and chromatography on and dendritic cells (DCs) (43), BK B1 receptor expression is a DEAE-Sepharose CL-6B column. SDS-PAGE analysis of the final prep- largely confined to inflamed tissues. Of note, the long-range ligand aration showed minor and major protein bands of 67 kDa (Mfa1) and 41 activity of BK/LBK (B R agonists) is prevented by the terminating kDa (FimA) (data not shown), respectively, indicating that a mixture of 2 major (FimA) and minor (Mfa1) Fim was obtained. The fimbrial prepara- action of host metallopeptidases, such as ACE (angiotensin-con- tions tested negative for endotoxin (Ͻ6 endotoxin U/mg protein), accord- verting ; Ref. 44) and TAFI (thrombin-activatable fibrino- ing to a quantitative Limulus amebocyte lysate assay (BioWhittaker). RgpB lysis inhibitor; Ref. 45). was obtained from the P. gingivalis HG66 strain culture fluid as previously In the past few years, immunological studies conducted in mice described (51). The purity of the enzyme was checked by SDS-PAGE. The infected by Trypanosoma cruzi, a parasitic protozoan equipped amount of active enzyme in purified RgpB was determined by titration using Phe-Pro-Arg-chloromethyl ketone (Bachem). The final prep- with a kinin-releasing (cruzipain), demonstrated aration of gingipain was assayed for possible contamination with LPS us- that conventional (immature) DCs sense the presence of this patho- ing the Limulus test and found to be negative (Ͻ6 endotoxin U). As a gen in peripheral or lymphoid tissues via triggering of B2R (46– cysteine proteinase, RgpB requires pretreatment with a reducing agent to 49). Once activated by kinins, immature CD11cϩ DCs develop become an active enzyme. Therefore, a stock solution of RgpB was 2-fold diluted in 0.2 M HEPES and 5 mM CaCl2 (pH 8.0), supplemented with 20 into the terminally differentiating APCs that emigrate to secondary mM cysteine, and incubated at 37°C for 15 min. The activated RgpB was lymphoid tissues where they prime naive T cells while steering then further diluted to the working concentrations with an appropriate TH1 polarization via the IL-12-dependent pathway (43, 46, 47). buffer. 3702 GINGIPAIN ROLE IN T CELL RESPONSES TO P. gingivalis

Edema assays and cytoplasmic color. Total neutrophils present in the gingival epithelium

Ϫ/Ϫ Ϫ/Ϫ and connective tissue proximal to the inferior incisors were determined in Male mice (BALB/c, C57BL/6, C57BL/6.TLR2 , or C57BL/6.B2R ) 40 sequential sections per mouse. Statistical differences between mean val- were injected intrabuccally (bottom of anterior mandibular vestibule) with ues were evaluated by ANOVA, and pairwise comparisons were done by ␮ ϫ 4 ⌬ 30 lof1 10 P. gingivalis W83 or with the Rgps mutant strain. the Tukey test. Where indicated, the mice were injected with P. gingivalis W83 pretreated (20 min at room temperature) with 10 ␮M D-Phe-Pro-Arg-chloromethyl Intravital digital microscopy of the hamster cheek pouch (HCP) ketone (Bachem) or with PBS as control. The buccal-lingual edema (vol- ume changes) was measured 3 h after bacteria inoculation, using a pa- Hamsters were anesthetized by i.p. injection of sodium pentobarbital sup- ␣ quimeter. The involvement of the kinin/B2R pathway in the development plemented with i.v. -chloralose (2.5% (w/v) solution in saline) through a of mucosal edema was investigated by pretreating the mice 1 h earlier with femoral vein catheter. A tracheal cannula (PE 190) was inserted to facilitate 100 ␮g/kg (s.c.) HOE-140 (Aventis). Depletion of polymorphonuclear neu- spontaneous breathing and the body temperature was maintained at 37°C trophils (PMN) was sought by pretreating (18 h) the intrabuccally infected by a heating pad monitored with a rectal thermistor. The HCP was prepared mice i.p. with 0.45 ml of a 1/10 dilution of rabbit antiserum to PMN and used for intravital microscopy as recently described (54). Briefly, the (Accurate Chemical Corporation) in PBS. As control groups, mice infected cheek pouch was mounted on a microscope stage and an area of ϳ1cm2 intrabuccally were pretreated with the same volume of normal rabbit serum was prepared for intravital microscopy observations of the microcircula- (46, 53). Paw edema induced by purified P. gingivalis molecules was de- tion. Thirty minutes after the preparation was completed, 100 mg of flu- termined 3 h after s.c injection of 25 ␮l of a PBS solution containing 7.5 orescein-labeled dextran per kilogram of body weight (FITC-dextran, 150 ng of LPS, alone or combined, to 10 nM (final concentration) RgpB. Where kDa molecular mass; TdB Consultancy) was injected i.v. as a macromo- indicated, we injected purified LPS combined with gingipain previously lecular tracer. The HCP was continuously superfused with a HEPES-bi- inactivated by 100 nM D-Phe-Pro-Arg-chloromethyl ketone. The paw carbonate-buffered saline solution (pH 7.4) at a constant rate of 5 ml/min. edema induced by LPS/gingipain or by living bacteria (controls) was ex- The microcirculation of the HCP was observed using an Axioskop 40 mi- ϫ ϫ pressed as volume differences between injected and contralateral paws and croscope, 4 objective and 10 ocular lens (Zeiss) equipped with appro- Downloaded from measured with the aid of a plethysmometer (46). priate filters (490/520 nm; FITC-dextran) for observations of fluorescence in epiluminescence. A digital camera, AxioCam HRc, and a computer with Kinins instruct type 1 cytokine production in the AxioVision 4.4 software program (Zeiss) were used for image analysis. P. gingivalis-infected mice Arteriolar diameters and total fluorescence were recorded in a representa- 2 ϳ 2 Ϫ/Ϫ Ϫ/Ϫ tive rectangular area (5 mm ) of the prepared HCP ( 1cm ). The recorded BALB/c, C57BL/6 WT, C57BL/6 TLR2 , and C57BL/6 B2R mice fluorescence at 30 min after FITC-dextran injection in each experiment was ϫ 4 were inoculated intrabuccally with 1 10 P. gingivalis W83, W83 pre- adjusted to 2000 fluorescent units for statistical reasons and arteriolar di- treated with the gingipain inhibitor D-Phe-Pro-Arg-chloromethyl ketone as ameter (range, 30–80 ␮m) at the same point in time was defined as 100%. http://www.jimmunol.org/ described above, or the ⌬ Rgps mutant strain. After 10 days, the infected ␮ Images were recorded at 5-min intervals during the entire experiment and mice were boosted intrabuccally with boiled Fim Ag (1 g/mouse). Seven fluorescence of the observed area (5 mm2) was used as a measure of plasma days later, the submandibular lymph node (LN) T cells were stimulated in leakage. In two series of experiments the bacteria suspensions (500 ␮lin vitro with boiled Fim Ag (100 ng/ml). As controls, Fim Ag was added to PBS) were applied twice at 35- to 40-min intervals during superfusion a culture of pooled LN T cells from normal mice. Culture supernatants ␥ arrest for 9 min. The first series of experiments consisted of two groups of were collected after 72 h, and IFN- and IL-17 were quantified by ELISA hamsters: 1) a control group (n ϭ 4) involving HCP exposed twice to W83 using purified and biotinylated Abs (R&D Systems). The effect of purified P. gingivalis; and 2) a test group involving bacteria application as de- P. gingivalis LPS and gingipain on the immune profile of OVA-specific T scribed above (positive control) followed by a second challenge in HCP cells was studied by immunizing BALB/c mice (pretreated or not with ␮ ␮ pretreated with 0.5 M HOE-140, added to the superfusion solution 5 min HOE-140 as described above) intrabuccally with 75 l of a formulation before the second bacteria application. Once fluorescence after the second ␮ by guest on September 26, 2021 containing OVA Ag (50 g/mouse) preabsorbed to aluminum hydroxide application had returned to preapplication levels, a continuous application (alum) at 5 mg/mouse, RgpB (10 nM), and LPS (7.5 ng). Ten days later, of BK (520) nM was made for 5 min to make sure that the effects of the immunized mice were boosted with OVA (25 ␮g/ml). Recall assays ␮ HOE-140 were prolonged. In the second series of experiments the HCP were performed by adding OVA (2.5 g/ml) to the cultures of LN T cells (n ϭ 5) was challenged with W83 P. gingivalis pretreated with Rgps in- isolated 7 days after a booster. Values are presented as picograms or nano- Ϯ hibitor for 10 min. A second application was made with the same inoculum grams of cytokine per milliliter (mean SD). Statistical differences be- of bacteria pretreated with the PBS diluent. Statistical evaluation was made tween mean values were evaluated by ANOVA, and pairwise comparisons by a paired t test comparing the responses of first bacteria application with were done by the Tukey test. those of the second. A p value of Ͻ0.05 was considered to indicate a Kininogen-dependent rescue of T cell responses in infected statistically significant difference. TLR2Ϫ/Ϫ and PMN-depleted BALB/c mice Results Ϫ/Ϫ TLR2 or PMN-depleted BALB/c mice infected intrabuccally with a Interstitial edema in infected mucosal tissues depends on suspension of 1 ϫ 104 P. gingivalis containing 10 ␮g of purified human Ϫ Ϫ kininogen (HK) per mouse (Calbiochem). As indicated, the TLR2 / or functional interplay between TLR2 and the kinin/B2R pathway PMN-depleted BALB/c mice challenged as described above were pre- Ϫ/Ϫ Given that oral mucosa is a natural niche for P. gingivalis colo- treated with HOE-140. As additional controls, the TLR2 or PMN-de- nization in humans, we first sought to determine whether the pres- pleted BALB/c mice were inoculated with a suspension of ⌬ Rgps strain supplemented with purified HK. After 10 days, mice were boosted with ence of this periodontal pathogen in mucosal tissues leads to the boiled Fim Ag (1 ␮g/mouse). Seven days later, submandibular LN T cells activation of the kinin system. This was approached by measuring were stimulated in vitro with boiled Fim Ag (100 ng/ml). Culture super- mucosal swelling in mice (BALB/c or C57BL/6) infected intra- ␥ natants were collected after 72 h, and IFN- and IL-17 were quantified by buccally with a low dose (104 cells) of WT P. gingivalis (W83). ELISA using purified and biotinylated Abs (R&D Systems). Values are presented as picograms or nanograms of cytokine per milliliter (mean Ϯ Measurements made 3 h postinfection (p.i.) indicated that P. gin- SD). Statistical differences between mean values were evaluated by givalis W83 evokes a potent gingival edema in both C57BL/6 (Fig. ANOVA, and pairwise comparisons were done by the Tukey test. 1) and BALB/c mice (data not shown). Notably, the mucosal Histology swelling evoked by P. gingivalis W83 was inhibited in mice pre- treated with the specific B2R antagonist HOE-140 (Fig. 1). Con- Maxillary tissues were harvested 48 h after P. gingivalis infection or after sonant with the pharmacological results, the WT bacteria did not injection of bacterial molecules (LPS and/or gingipain) into the mandibular Ϫ Ϫ ϩ ϩ Ϫ Ϫ / anterior vestibule of BALB/c, TLR2 / (C57BL/6 WT), TLR2 / , induce significant mucosal swelling in C57BL/6.B2R mice ϩ/ϩ Ϫ/Ϫ B2R (C57BL/6 WT), or B2R mice. Tissues were fixed overnight in (Fig. 1). Next, we asked whether the WT bacteria relied on gin- 10% neutral buffered formalin, decalcified in CalExII (Fisher Scientific) for gipain to generate kinins in mucosal tissues. First, we found that P. 72 h, and dehydrated in 20% ethanol for 1 h, 50% ethanol for 1 h, and 70% gingivalis W83 pretreated with the Rgps-specific inhibitor was un- ethanol for 1 h. Samples were embedded in paraffin and serially sliced into 0.2-␮m sagittal sections and stained with H&E. Sections were analyzed able to induce significant mucosal swelling (Fig. 1). Second, we under oil immersion at ϫ1000 original magnification. Positive identifica- found that intrabuccal injection of the mutant strain ⌬ Rgps (a tion of tissue neutrophils was determined by matching nuclear morphology double knockout mutant strain with deleted RgpA and RgpB The Journal of Immunology 3703

lation at 10 and 15 min ( p Ͻ 0.05) (Fig. 2A). Interestingly, HOE-140 did not block the early leakage response evoked by the W83 strain (Fig. 2A). Of note, the application of exogenous BK as an internal

control indicated that B2R expression on the HCP vascular beds is preserved at the end of these assays (Fig. 2A). Combined, these results suggested that the initial increase in vascular permeability evoked by

W83 is driven by kinin/B2R-independent mechanisms. Noteworthily, however, we found that the plasma leakage response (10–20 min) evoked by the second bacteria challenge was significantly reduced by Ͻ the B2R antagonist ( p 0.05). Given indications that the kinin/B2R pathway was activated upon second bacterial challenge, we then asked whether gingipain was involved in this process. Indeed, we found that plasma leakage evoked by the second topical application of W83 (peaking at 10–30 min) was substantially reduced when we instead exposed the HCP to W83 pretreated with the Rgps inhibitor ( p Ͻ 0.05) (Fig. 2B, lower panel). Intriguingly, however, the latter treatment did not cancel the vasodilatory responses (Fig. 2B, upper panel), thus suggesting that gingipain enzyme activity is not essential

for induction of vasodilatation. Collectively, the intravital microscopy Downloaded from studies in the HCP suggest that early vascular permeability increases

evoked by P. gingivalis W83 are coordinated by kinin/B2R-indepen- dent mechanisms. However, following the onset of inflammation, the initial plasma leakage is rapidly intensified through gingipain-depen- dent generation of vasoactive kinins (B R agonists). FIGURE 1. Mucosal edema induced by P. gingivalis depends on the 2 http://www.jimmunol.org/ functional interplay between TR2, neutrophils, and the gingipain/B2R path- Activation of the TLR2/B2R axis by P. gingivalis promotes way. The graphs depict the intensity of mucosal edema (buccal-lingual) in neutrophil recruitment to the inflamed mucosal tissues C57BL/6 WT (TLR2ϩ/ϩ) vs C57BL/6.TLR2Ϫ/Ϫ and C57BL/6 WT ϩ/ϩ Ϫ/Ϫ (B2R ) vs C57BL/6.B2R mice that were inoculated 3 h earlier (man- Previous analysis of the dynamics of inflammation elicited by T. dibular anterior vestibule) with 30 ␮l of either P. gingivalis W83 WT cruzi suggested that neutrophils link TLR2-dependent recognition strain, the Rgps-deficient mutant ⌬ Rgps, or WT W83 bacteria pretreated of microbial signatures to the kinin proteolytic cascade (55). In this with the Rgps inhibitor. Where indicated, the mice were pretreated with study we asked whether similar mechanisms may operate in W83- HOE-140 before injection of the bacteria. A separate group of mice infected mucosal tissues. To this end, we injected P. gingivalis (C57BL/6 WT) were pretreated (18 h before bacteria injection) i.p. with W83 intrabuccally in C57BL/6 mice that were previously (18 h

0.45 ml of a 1/10 dilution in PBS of rabbit antiserum to PMN or an equiv- by guest on September 26, 2021 earlier) immunodepleted of neutrophils. Analysis of gingival alent volume of normal rabbit serum (control). The results (means Ϯ SD) are representative of three independent experiments (n ϭ 5 mice/group). swelling (3 h p.i.) revealed that the inflammatory edema was nul- Statistical differences between mean values were evaluated by ANOVA, lified in neutrophil-depleted mice (data not shown), in contrast to and pairwise comparisons, indicated by the lowercase letters, were done by the intense swelling reactions observed in control animals (pre- p Ͻ 0.01). treated with nonimmune serum). We then sought to characterize ,ء) the Tukey test the inflammatory infiltrates recruited to the gingival tissues (Fig. 3). Morphometric analysis revealed the presence of prominent genes) did not induce significant mucosal swelling in C57BL/6 neutrophil infiltrates in mucosal tissues of infected C57BL/6 ϩ/ϩ Ϫ/Ϫ Ϫ/Ϫ (Fig. 1) or BALB/c mice (data not shown). Collectively, these TLR2 mice, but not in TLR2 or C5BL/6.B2R mice results suggested that bacterial competence to induce mucosal (Fig. 3). Moreover, there was no significant neutrophil infiltration swelling via activation of the kinin/B2R pathway depends on the in mucosal tissues of C57BL/6 mice injected intrabuccally with enzymatic activity of gingipain. either the ⌬ Rgps bacterial strain (Fig. 3A, upper panel)orWT Given indications that activation of TLR2 is required for kinin bacteria pretreated with the gingipain inhibitor (data not shown). system activation in the mouse model of T. cruzi infection (46), we Altogether, these results strongly suggested that the W83-induced then asked whether mucosal swelling evoked by P. gingivalis is infiltration of neutrophils into mucosal tissues depends on the in- 3 likewise subordinated to TLR2 signaling by microbial “signa- terplay between TLR2 and the gingipain kinin/B2R pathway. tures”. Consistent with this premise, our results revealed that P. gingivalis W83 did not induce appreciable edema in C57.BL/ LPS and gingipain, acting synergistically, drive Ϫ Ϫ 6.TLR2 / mice (Fig. 1). Of note, these animals developed a neutrophil-dependent edema via the TLR2/B2R pathway prominent edema upon injection of synthetic BK, thus confirming Seeking for proof of concept in favor of our working hypothesis, Ϫ/Ϫ that B2R function is intact in the TLR2 deficient mice (46). we then asked whether the proinflammatory phenotype of P. gin- Considering that swelling measurements in the buccal mucosa are givalis W83 could be recapitulated by injecting mice with LPS, an made at relatively late time points (3 h), we resorted to intravital atypical TLR2 ligand (16), along with the kinin-releasing protease, microscopy in the HCP to investigate the early-phase microvascular i.e., gingipain. Rather than injecting LPS/gingipain in the anterior responses evoked by the periodontal pathogen. Our results showed mandibular vestibule, the purified bacterial molecules were inoc- that topically applied P. gingivalis W83 induced an immediate arte- ulated in the more sensitive paw tissue (Fig. 4). Control experi- riolar dilation (Fig. 2A, upper panel) accompanied by increased ments performed in BALB/c mice confirmed that the living W83 plasma leakage (Fig. 2A, lower panel) peaking at ϳ10 min, both of bacteria potently induced paw edema responses via the gingipain/ which were resolved after 35–40 min. A second challenge with the B2R pathway (Fig. 4). In contrast, there was no detectable footpad WT W83 strain yielded vascular responses of similar magnitude. Pre- swelling in BALB/c mice injected with either LPS or activated treatment of HCP with HOE-140 resulted in a significant smaller di- gingipain alone (Fig. 4). Strikingly, however, mice injected with 3704 GINGIPAIN ROLE IN T CELL RESPONSES TO P. gingivalis Downloaded from http://www.jimmunol.org/

FIGURE 2. Intravital microscopy analysis of early-phase vascular permeability increases elicited by topically applied P. gingivalis. A, Upper panel, Diameter changes (means Ϯ SEM) in arterioles in two groups of HCP (n ϭ 5) topically exposed to W83 P. gingivalis. Control experiments were performed by adding HOE-140 to one group of hamsters. Note that there was a significant smaller diameter increase as compared with the untreated control group p Ͻ 0.05. Lower panel, The graph describes plasma leakage (FITC-dextran) responses (means Ϯ SEM) for two ,ء ;and 15 min after bacteria application 10 groups of hamsters. After local application of HOE-140 to one group (see second bacteria application) there was significantly less plasma leakage at 10, p Ͻ 0.05. B, Upper panel, Diameter changes (means Ϯ SEM) in HCP arterioles in one ,ء ,and 20 min as compared with the untreated control group ,15 by guest on September 26, 2021 group of hamsters (n ϭ 5) receiving bacteria applications with a 40-min interval. The first application was made with the P. gingivalis W83 (Bact 1) pretreated in vitro with Rgps inhibitor. There was a significant difference (p Ͻ 0.05) only in the arteriolar diameter at 10 min after the application of W83 P. gingivalis (Bact 1) plus inhibitor as compared with 10 min after the application of W83 without Rgps inhibitor (Bact 2). Lower panel, Vascular permeability changes (means Ϯ SEM) recorded in two groups of hamsters. There were significant differences in plasma leakage (fluorescence) induced by p Ͻ 0.05). Plasma leakage increase after application of W83 (Bact 1) plus ,ء) the first and the second application of bacteria at 10, 15, 20, 25, and 30 min Rgps inhibitor was 64% of the control response induced by the untreated W83 (Bact 2). Statistical evaluation was made by a paired t test comparing the responses of the first bacteria application with those of the second. the combination of LPS and activated gingipain developed a strate that LPS and gingipain, acting synergistically, can elicit potent paw edema (Fig. 4A). Of note, these proinflammatory edema and gingivitis through mechanisms involving coopera- effects were blocked either by pretreating BALB/c mice with tive activation of TLR2/neutrophils and B2R. HOE-140 or by preincubating purified RgpB with the specific inhibitor before mixing it to the LPS sample (Fig. 4A). Consis- BALB/c mice orally infected by P. gingivalis develop tent with a role for neutrophils in this process, neutrophil-de- IFN-␥ and IL-17 producing anti-Fim T cells in 3 pleted BALB/c mice failed to develop significant paw edema gingipain kinin/B2R-dependent manner upon injection of LPS/gingipain, whereas control mice pre- In the next series of experiments we asked whether kinins gener- treated with normal serum showed overt swelling under the ated in W83-infected mucosal tissues coordinate adaptive immu- same condition (Fig. 4A). Similarly to data shown for P. gin- nity via activation of the B2R pathway. To this end, we inoculated givalis W83, the injection of LPS/gingipain evoked potent paw BALB/c mice intrabuccally with P. gingivalis W83 or with the Ϫ/Ϫ Ϫ/Ϫ edema in C57BL/6 mice, but not in TLR2 or B2R mutant mutant bacterial strain ⌬ Rgps. Ten days after infection, both mice (Fig. 4, B and C). Collectively, these results indicate that groups of BALB/c mice were boosted with Fim Ag and the T cells purified LPS and gingipain (RgpB), acting as a microbial-de- were isolated 7 days later from submandibular draining LNs. The rived TLR2 ligand and a kinin-releasing protease, respectively, results from recall assays performed with Fim Ag showed that are able to recapitulate the proinflammatory phenotype of P. experienced T cells from W83-infected BALB/c mice produced gingivalis. Noteworthily, the intrabuccal injection of LPS/gin- ␥ high amounts of IFN- as well as the TH17 cytokine, IL-17 (Fig. gipain also elicited potent neutrophil infiltration in mucosal tis- 5). In contrast, Fim-specific T cells from infected mice pretreated Ϫ/Ϫ sues of C57BL/6 mice but not in those of TLR2 deficient with a single dose of HOE-140 showed drastically reduced IFN-␥ 4 mice (supplemental Fig. S1). Altogether, these results demon- and IL-17 responses (Fig. 5). In line with the concept that induc- tion of adaptive immunity is critically dependent on gingipain- 4 The online version of this article contains supplemental material. mediated generation of kinins in mucosal tissues, we found that The Journal of Immunology 3705 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. TLR2/B2R “cross-talk” drives bacteria-induced gingivitis. C57BL/6 WT mice pretreated or not pretreated with HOE-140 (as indi- cated) were orally infected (buccal vestibule) with either WT P. gingivalis W83, the ⌬ Rgps strain, or P. gingivalis W83 pretreated with the Rgps ϩ/ϩ ϩ/ϩ inhibitor. In a second scheme, C57BL/6 WT (TLR2 and B2R ), Ϫ/Ϫ Ϫ/Ϫ TLR2 , and B2R mice were infected as described above with WT P. gingivalis W83. Mice were killed 48 h after inoculation and the histological sections of gingival tissues were obtained and stained with H&E. A, His- togram analysis showing the numbers of neutrophils in representative gin- FIGURE 4. Purified P. gingivalis LPS and gingipain induce paw edema in ␮ gival sections. At least 80 sections per condition were examined for neu- a TLR2/B2R-dependent manner. Edema was induced by the injection of 25 l trophils by oil immersion microscopy at ϫ1000 original magnification. of PBS containing 7.5 ng of purified P. gingivalis LPS alone or combined with Statistical differences between mean values were evaluated by ANOVA, 10 nM (final concentration) RgpB preactivated with 10 mM cysteine into the ϩ/ϩ Ϫ/Ϫ and pairwise comparisons, indicated by the lowercase letters, were done by paws of BALB/c (A), C57BL/6 (TLR2 ) vs C57BL/6.TLR2 (B), and ϩ/ϩ Ϫ/Ϫ the Tukey test (p Ͻ 0.05). Arrows indicate the neutrophil infiltrates. B, C57BL/6 (B2R ) vs C57BL/6.B2R (C) mice. PBS was injected in the Corresponding images acquired with a Zeiss Axio Imager microscope contralateral paw. Controls are done with gingipain irreversibly inactivated by system. the Rgps inhibitor. Mice were pretreated or not pretreated with HOE-140 1 h before the injection. Volume differences were determined after 3 h p.i. The results (means Ϯ SD) are representative of three independent experiments BALB/c mice infected with ⌬ Rgps or with W83 bacteria pre- (n ϭ 5 mice/group). Statistical differences between mean values were evalu- treated with the gingipain inhibitor failed to produce IFN-␥ and ated by ANOVA, and pairwise comparisons, indicated by the lowercase let- .(p Ͻ 0.01 ,ء) IL-17 (Fig. 5). Of note, control experiments indicated there was no ters, were done by the Tukey test significant secretion of IFN-␥ and IL-17 cytokines when we added Fim Ag to T cells derived from LNs of noninfected BALB/c mice (data not shown). Collectively, these results suggested that gingi- phenotype (induction of mucosal edema and neutrophil infiltra- pain-mediated generation of kinins in the inflamed mucosa of tion) of P. gingivalis W83. In this study we checked whether mice W83-infected BALB/c mice drives production of IFN-␥- and IL- immunized with a conventional immunogen (OVA) in the pres-

17-producing, Fim-experienced T cells in B2R-dependent manner. ence of purified LPS and active gingipain could generate IFN-␥- and IL-17-producing T cells in a kinin/B R-dependent manner, ␥ 2 Purified LPS and gingipain, acting synergistically, induce IFN- - hence reproducing effects ascribed to P. gingivalis W83 infection. and IL-17-producing T cells in kinin/B2R-dependent manner To this end, we immunized BALB/c mice intrabuccally with alum- Earlier in this work, we presented evidence that purified LPS com- based emulsions containing OVA immunogen supplemented (or bined to active gingipain could recapitulate the proinflammatory not) with purified LPS/gingipain (RgpB). At day 10, the mice were 3706 GINGIPAIN ROLE IN T CELL RESPONSES TO P. gingivalis Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Mice immunized intramucosally with OVA/alum supple- FIGURE 5. Functional profile of Fim-experienced T cells isolated from mented with LPS and gingipain generate T 1 responses via the B R pathway. P. H 2 submandibular draining LNs of BALB/c mice infected intrabuccally with BALB/c mice were immunized intrabucally with OVA Ag (25 ␮g/mouse) gingivalis . BALB/c mice were pretreated or not pretreated with HOE-140 1 h preabsorbed into alum, with or without supplementation with W83-derived P. gingivalis before challenge in the mandibular anterior vestibule with WT, LPS (7.5 ng) and RgpB preactivated with 10 mM cysteine. Separate groups of WT pretreated with D-Phe-Pro-Arg-chloromethyl ketone (gingipain inhibitor) mice were pretreated or not with a s.c. injection of HOE-140 1 h before im- ⌬ ␮ or the Rgps strain. Ten days later, mice were boosted with Fim Ag (1 g/ munization. One week later, mice were boosted with OVA Ag (10 ␮g/mice) mouse). Submandibular LN T cells were harvested 7 days after the booster and at the same site. T cells were harvested from submandibular LNs 7 days after were stimulated with medium alone or with Fim Ag (100 ng/ml) for 72 h at the booster and stimulated with medium alone or OVA (1 ␮g/mice) for 72 h ␥ A B 37°C before titers of IFN- ( ) and IL-17 ( ) in the culture supernatants were at 37°C. Titers of IFN-␥ (A) or IL-17 (B) in the culture supernatants were determined by ELISA. Results are representative of three independent exper- determined by ELISA. Results are representative of three independent exper- n ϭ iments with similar results ( 5 mice/group). Differences were analyzed by iments with similar results. Statistical differences between mean values were ANOVA and pairwise comparisons, indicated by the lowercase letters, were evaluated by ANOVA, and pairwise comparisons, indicated by the lowercase p Ͻ ء .(p Ͻ 0.01 ,ء) done by the Tukey test ( , 0.05). letters, were done by the Tukey test boosted with soluble OVA (same immunization site). Seven days that addition of the Rgps inhibitor to the alum/OVA immunogen later, the submandibular draining LNs were recovered and the (s.c. injected BALB/c mice) did not interfere with the magnitude

OVA-specific recall responses were analyzed (Fig. 6). The results of the prototypical TH2 polarized anti-OVA responses observed in showed that IFN-␥ and IL-17 were vigorously secreted by OVA- this inbred strain (data not shown). specific T cells from mice immunized with alum-based OVA com- Purified kininogen reconstitutes the type 1 deficient responses of bined with both LPS and gingipain. Importantly, OVA-specific T Ϫ/Ϫ cells were no longer able to secrete IFN-␥ and IL-17 if the mice TLR2 mice immunized as described above were pretreated with HOE-140 In this study we tested the hypothesis that the availability of high (Fig. 6). Along similar lines, the production of IFN-␥ and IL-17 by levels of kininogens (i.e., gingipain substrate) in peripheral tissues OVA-experienced T cells was significantly diminished when the is a limiting factor governing the cytokine profile of Fim-specific enzymatically active gingipain RgbB present in the alum-based effector T cells. Recall assays performed with Fim Ag indicate that OVA/LPS formulation was replaced by gingipain inactivated by submandibular T cells from W83-infected TLR2ϩ/ϩ mice (posi- the Rgps inhibitor (Fig. 6). Of note, control experiments showed tive controls) vigorously up-regulated IFN-␥, whereas HOE-140, The Journal of Immunology 3707 Downloaded from

FIGURE 7. Purified HK corrects the adaptive immune dysfunction of Fim-specific T cells from TLR2Ϫ/Ϫ mice infected intramucosally by P. gingivalis. C57BL/6 WT (TLR2ϩ/ϩ) mice were pretreated or not pretreated with HOE-140, as indicated, before infection (bottom of mandibular ante- rior vestibule) with wild-type P. gingivalis W83 or the ⌬ Rgps strain. http://www.jimmunol.org/ Where indicated, mice were infected with the WT bacteria pretreated with the gingipain inhibitor. In a second scheme, TLR2Ϫ/Ϫ mice pretreated or not pretreated with HOE-140 as indicated were orally infected with sus- pensions of P. gingivalis W83 (WT or ⌬ Rgps strain) supplemented with purified HK. After 10 days, mice were boosted with Fim Ag (1 ␮g/mouse). Submandibular LN T cells were recovered and stimulated with Fim Ag (100 ng/ml) in vitro. Culture supernatants were collected after 72 h and IFN-␥ was quantified by ELISA. Statistical differences between mean val- ues were evaluated by ANOVA, and pairwise comparisons, indicated by by guest on September 26, 2021 .(p Ͻ 0.05 ,ء) the lowercase letters, were done by the Tukey test as predicted, canceled the type 1 response (Fig. 7). Similarly, TLR2ϩ/ϩ mice did not generate IFN-␥-producing anti-Fim T cells following injection of P. gingivalis W83 pretreated with the Rgps inhibitor (Fig. 7). In contrast, we found a marked reduction of IFN-␥ production by Fim-experienced T cells from W83-infected TLR2Ϫ/Ϫ mice (Fig. 7). We then checked whether the combined FIGURE 8. Purified HK reconstitutes the deficient generation of IFN- ␥-producing and IL-17 producing Fim-specific T cells in neutrophil-de- injection of P. gingivalis W83 and HK into the mandibular anterior Ϫ/Ϫ pleted BALB/c mice infected intrabuccally by P. gingivalis. Levels of vestibule of TLR2 animals would rescue the otherwise defi- IFN-␥ (A) and IL-17 (B) secreted by submandibular LN Ag-specific T cells cient type 1 responses of these transgenic mice. Indeed, anti-Fim T isolated from BALB/c mice orally infected (bottom of the mandibular an- Ϫ/Ϫ cells isolated from TLR2 mice that received HK at the time of terior vestibule) with either WT P. gingivalis W83 or the ⌬ Rgps strain. bacterial challenge robustly secreted IFN-␥, and these effects were Where indicated, mice were pretreated with HOE-140 1 h before the in- canceled by HOE-140 (Fig. 7). Collectively, these results indicate jection. A separate group of mice were depleted of circulating neutrophils that exogenous HK bypassed the requirement for TLR2-driven in- 18 h before infection i.p. with 0.45 ml of a 1/10 dilution in PBS of rabbit flux of plasma borne kininogens. In other words, exogenous HK antiserum to PMN or an equivalent volume of normal rabbit serum (con- underwent proteolytic processing in a gingipain-dependent manner trol). PMN-depleted mice were either challenged with P. gingivalis W83 or ⌬ liberating the B R agonist, which then acted as a type 1-directing Rgps strain. In a second scheme, PMN-depleted mice were infected with 2 P. gingivalis (WT and ⌬ Rgps strains) combined with purified HK pre- adjuvant, thus restoring the immune dysfunction of TLR2-deficient treated or not pretreated with HOE-140, as indicated. After 10 days, the mice. Next, we verified whether the exogenous provision of HK to infected mice were boosted with Fim Ag (1 ␮g/mouse). Submandibular LN PMN-depleted BALB/c mice would also bypass the requirement T cells were recovered and stimulated with Fim Ag (100 ng/ml) in vitro. of neutrophils for kinin/B2R-dependent type 1 induction. Infected Culture supernatants were collected after 72 h and IFN-␥ and IL-17 were BALB/c-mice pretreated with nonspecific rabbit serum displayed quantified by ELISA. Statistical differences between mean values were slightly reduced IFN-␥ and IL-17 production by anti-Fim T cells. evaluated by ANOVA, and pairwise comparisons, indicated by the lower- .(p Ͻ 0.01 ,ء) In contrast with these controls, Fim-stimulated T cells isolated case letters, were done by the Tukey test from PMN-depleted, infected BALB/c mice or from BALB/c mice that were infected with the ⌬ Rgps strain did not secrete detectable levels of IFN-␥ or IL-17 (Fig. 8). Moreover, HOE-140 brought (Fig. 8). Notably, the deficient adaptive responses of PMN-de- about a partial, albeit significant reduction of IFN-␥ and IL-17 pleted BALB/c mice were fully reconstituted when these animals production by anti-Fim T cells from W83-infected BALB/c mice were injected with P. gingivalis W83 combined with exogenous 3708 GINGIPAIN ROLE IN T CELL RESPONSES TO P. gingivalis

HK (Fig. 8). Importantly, the combined injection of purified HK in either TLR2Ϫ/Ϫ or neutrophil-depleted B6 mice (Figs. 7 and 8). and the P. gingivalis ⌬ Rgps mutant strain failed to induce detect- These experiments indicated that the direct delivery of exogenous able IFN-␥ or IL-17 levels by Fim-stimulated T cells. Taken to- kininogens (gingipain substrate) into the peripheral sites of infec- gether, these results suggest that proteolytic generation of the kinin tion bypassed the requirement for plasma influx via the TLR2/ danger signal in infected mucosal tissues is preceded by the ex- PMN-dependent pathway. In other words, we may infer that travascular accumulation of plasma-borne kininogens, a response plasma leakage, a vascular reaction orchestrated through bacteria- driven by bacterial-induced activation of TLR2/neutrophils at induced activation of TLR2/neutrophils, is a limiting step control- early stages of intrabuccal infection. Once released at high levels, ling the extent of gingipain-mediated release of kinin “danger” kinins induce IFN-␥-producing, Fim-specific T cells (in C7BL/6 signals in mucosal tissues. and BALB/c mice) and IL-17-producing Fim-specific T cells (in Although we cannot rule out the possibility that innate sentinel

BALB/c mice) in a B2R-dependent manner. cells might sense P. gingivalis via TLR2/Fim (20), in this study we present evidences that the proinflammatory phenotype of P. gin- Discussion givalis W83 is recapitulated by injecting mice with only two pu- Confronted with a highly diversified microbial flora in the oral and rified virulence factors, i.e., LPS and gingipain (enzymatically ac- digestive mucosae, the immune system is able to discriminate tive). This conclusion was based on the following evidence: 1) commensal microbes from pathogens. Although the repeated ex- LPS/gingipain induces a potent buccal-lingual edema in B6 mice posure of the innate immune system to the commensal flora usu- via the TLR2/B2R pathway; and 2) BALB/c mice injected (intra- ally leads to immunological tolerance, the detection of danger cues buccally) with OVA/alum formulations containing purified LPS displayed by pathogens is translated into inflammation and adap- and gingipain-developed, IFN-␥-producing anti-OVA T cells and Downloaded from tive immunity (56). In the present work, we demonstrate that the IL-17-producing anti-OVA T cells in B2R-dependent manner. functional profile of Fim-specific effector T cells in mice infected A large body of the experimental evidence presented in this intramucosally by P. gingivalis W83 is coordinated by TLR2/B2R, article support the hypothesis that this Gram-negative bacteria in- an innate pathway forged by the cooperative signaling of two dif- duces edema by sequentially activating the TLR2/B2R axis via ferent types of danger signals: LPS, an atypical TLR2 ligand ex- LPS (TLR2 ligand) and kinins (B2R agonists) released by gingi- pressed by P. gingivalis, and bradykinin, a proinflammatory en- pain. In general terms, the mechanistic principles outlined here are http://www.jimmunol.org/ dogenous peptide released from plasma-borne kininogens through reminiscent of those described in studies of mice s.c. infected with the proteolytic activity of gingipain. T. cruzi (46). According to our working hypothesis (Fig. 9), P. By developing an infection model based on low dose intrabuccal gingivalis initiates mucosal inflammation through the activation of injection of the periodontal bacteria, we were able to demonstrate TLR2 expressed by resident cells (epithelial, macrophages, and that blockade of the kinin/B2R pathway at the onset of inflamma- mast cells). Following up-regulated secretion of chemokines, neu- tion modifies the cytokine profiles of Fim-experienced T cells iso- trophils adhere to the activated endothelium and drive the initial lated (17 days p.i.) from the submandibular draining LNs. While influx of plasma (including kininogens) into extravascular tissues. admitting that other Ag specificities may likely contribute to the Acting further downstream, P. gingivalis may generate high levels pool of antibacterial effector T cells isolated from submandibular of vasoactive kinins through the activity of gingipain, thus further by guest on September 26, 2021 draining LNs, we chose to use Fim Ag as the readout in recall amplifying plasma leakage. This sequential model is consistent assays because fimbriated strains of P. gingivalis (rather than non- with data presented in the current work, showing that TLR2-defi- fimbriated strains) are frequently detected in deep periodontal cient mice and neutrophil-depleted B6 mice failed to develop ki- pockets and in sites of severe periodontal attachment loss (57). nin/B2R-dependent gingivitis (Fig. 1). This result, combined with Experiments performed in BALB/c mice infected with P. gingi- evidence from intravital microscopy studies showing that topically valis W83 (WT) revealed that the bacteria evoked a strong buccal- applied P. gingivalis W83 does not immediately activate the kinin/ lingual edema via the B2R pathway (i.e., blocked by HOE-140 or B2R pathway in the HCP microvascular beds but does so after a absent in B2R-deficient mice) and provided evidence that B2R- second consecutive application of the pathogen, supports the view driven mucosal inflammation leads to the accumulation of IFN-␥- that the activation of TLR2/neutrophils at the upstream end of the producing, Fim-specific T cells and to IL-17-producing Fim T cells inflammatory process might allow for the accumulation of plasma- in submandibular draining LNs. We also showed that mice chal- borne kininogens (i.e., gingipain substrate) into extravascular tis- lenged with W83 pretreated with an irreversible gingipain inhibitor sues. In view of complex structural requirements for TLR4 vs or inoculated with a gingipain-deficient P. gingivalis W83 mutant TLR2 signaling by P. gingivalis LPS (16), additional studies in ⌬ ( Rgps) not only failed to develop B2R-dependent gingivitis, but TLR4-deficient mice will be required to verify whether TLR4 may the Fim-experienced T cells present in draining LNs did not se- likewise propel activation of the kinin proteolytic system. What- crete IL-17 (BALB/c) or IFN-␥ (BALB/c and B6 mice). Com- ever the structural basis for LPS triggering of TLR4/TLR2, our 3 bined, these data linked activation of the gingipain kinin/B2R study showed that the intrabuccal injection of purified HK along pathway at the onset of mucosal infection to the shaping of the T with P. gingivalis W83 (but not with the gingipain-deficient cell branch of (adaptive) immunity. Complimentary experiments ⌬ Rgps strain) rescued the otherwise deficient type 1 responses of performed with W83-infected B6.TLR2Ϫ/Ϫ and with neutrophil- TLR2Ϫ/Ϫ or neutrophil-depleted mice. These data are in agree- depleted B6 mice indicated that their phenotypes were reminiscent ment with the concept that plasma-borne kininogens undergo pro- Ϫ/Ϫ of that displayed by B2R mice, i.e., these mice did not develop teolytic processing by gingipain at the downstream end of the in- significant gingivitis and the draining LN T cells from these mice flammatory cascade initiated by TLR2 ligands. Once liberated in ␥ failed to secrete IFN- upon ex vivo stimulation with Fim. Re- mucosal tissues, kinins induce TH1- and/or TH17-polarizing cyto- Ϫ/Ϫ markably, the immune dysfunction of B6.TLR2 and neutro- kines by APCs in a B2R-dependent manner. phil-depleted B6 mice was corrected when these animals were in- An unresolved issue in the present study concerns the identity of jected intramucosally with a suspension of P. gingivalis W83 the APCs that sense kinins released by gingipain in the inflamed supplemented with purified HK (Figs. 7 and 8). In contrast, the mucosal tissues (58, 59). For example, it remains to be determined addition of purified HK to a suspension of gingipain-deficient whether B2R-dependent induction of TH1-polarizing cytokines ⌬ ␤ ( Rgps) strain failed to rescue the Fim-specific type 1 responses (IL-12p70) (43, 47) vs TH17-instructive signals (IL-6, TGF- , and The Journal of Immunology 3709 Downloaded from http://www.jimmunol.org/

FIGURE 9. Trans-cellular TLR2/B2R “cross-talk” drives proteolytic generation of immunostimulatory bradykinin in extravascular mucosal tissues. On the left side of the panel P. gingivalis W83 (injected in the bottom of mandibular anterior vestibule) stimulates TLR2 expressed by hitherto uncharacterized resident sentinel cells (e.g., gingival epithelial cells, macrophages, and mast cells). This innate recognition response translates into the secretion of chemokines/cytokines, which then rapidly amplify inflammation through the activation of neutrophils/endothelium. Owing to increased vascular perme- ability, plasma proteins extravasate, leading to the accumulation of blood-borne kininogens in extravascular tissues (center of panel). Further downstream, the cysteine protease gingipain proteolytically liberates the vasoactive kinin moiety from kininogens. Plasma leakage is fueled through reiterative cycles of B2R-dependent triggering of the endothelium by the vasoactive kinins. Based on analysis of the immunological roles of kinins released by T. cruzi (46, 48, 49), we hypothesize that immature (myeloid) DCs patrolling the inflamed mucosa (see rectangle) are activated via the kinin/B2R pathway. After ␥ migrating to submandibular draining LNs, the Fim-bearing DCs prime naive T cells while simultaneously instructing their development into IFN- - by guest on September 26, 2021 producing effector T cells (in BALB/c and C57BL/6 mice) and IL-17-producing T cells (BALB/c mice).

IL-23; Refs. 5–9) is differentially regulated in BALB/c and Fim-specific T cells in mice infected intrabuccally with P. C57BL/6 mice, perhaps reflecting in this context differences in the gingivalis. activation threshold of B2R expressed by DCs residing/recruited to Despite the variety of animal models and assay systems de- inflamed mucosal tissues (58, 59). Whether involving specialized scribed in the literature, several studies suggest that TLR2 activa- tissue DC subsets, such as Langerhans cells and dermal interstitial tion by P. gingivalis might serve the needs of the periodontal cells, the sampling and processing of Fim Ags by intramucosal pathogen, either because TLR2 signaling reduces bacterial uptake APCs may involve direct endocytic uptake of W83 P. gingivalis by phagocytes (18, 19) and/or because TLR2 up-regulates CR3, a and/or may depend on APC-mediated internalization of apoptotic surface receptor that down-modulates microbicidal responses of (bacterial-loaded) neutrophils (60). Worthy of note in this context, mononuclear phagocytes (20, 21). In contrast, there is evidence it has been reported that the internalization of apoptotic neutrophils that TLR2 signaling might benefit the host immune response; for by macrophages and DCs leads to decreased secretion of IL-23, a example, it was reported that P. gingivalis induced TLR2-depen- cytokine controlling IL-17 production by different subsets of T dent secretion of proinflammatory cytokines (e.g., IL-1␤, TNF-␣, cells (60). In view of these findings, it is conceivable that IL-23/ and IFN-␥) in s.c. inserted mouse chambers (63). Somewhat sur- IL-17-driven granulopoiesis might be differentially regulated in prisingly, these authors also noted that alveolar bone loss was at- BALB/c and C57BL/6 mice infected intrabuccally by P. gingivalis tenuated in TLR2-deficient mice (infected for 6 wk) (63), thus (61). Although speculative, another possible explanation for the implying that excessive activation of TLR2 may somehow stimu- discrepant immune profiles observed in W83-infected BALB/c and late the activity of bone-resorbing osteoclasts. In another report, C57BL/6 mice may relate to the fact that bacteria-derived mu- TLR2 of human gingival epithelial cells was shown to cooperate ramyldipeptides are sensed by NOD2 (nucleotide oligomerization with the T cell cytokine IL-17 (64). Although these findings raise domain 2), an intracellular pattern recognition receptor of human the possibility that mucosal inflammation elicited by P. gingivalis

DCs whose activation leads to induction of the TH17-polarizing LPS and/or by alternative TLR2 ligands might up-regulate TH17- cytokines IL-23 and IL-1 (62). Adding another level of complexity dependent bone resorption via the kinin/B2R pathway, the infec- to this problem, it was recently reported that gingipain, acting tion conditions used in the present study did not lead to persistent synergistically with ligands for TLR2 and NOD1/2 ligands, up- infection (data not shown), thus precluding an assessment of the regulates the production of IL-6 (a cytokine implicated in TH17 role of IL-17-producing, Fim-specific effector T cells in chronic development) by macrophages through the signaling of protease- periodontitis. Modified versions of our infection model may help activated receptors (37). Admittedly, additional studies may clarify to clarify whether sustained activation of the TLR2/B2R axis in whether activation of NOD1/2 might shape the cytokine profile of W83-infected-BALB/c and other mice strains may promote the 3710 GINGIPAIN ROLE IN T CELL RESPONSES TO P. gingivalis

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