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Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva

The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1β neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.

We have reported that kinins may play important roles in periodontitis 7 . Accordingly, B 1 and B 2 receptors are expressed on osteoblasts and fibroblasts and activation of these receptors causes enhanced bone resorption mediated by increased prostaglandin E 2 (PGE 2 ) formation in both cell types and enhanced expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts 3,8,9 . Interestingly, P. gingivalis expresses an arginine specific cysteine proteinase (Arg-gingipain-1/RGP-1) that can release kinins from kininogens 10 , facilitated by components of the kallikrein-kinin system binding to gingipains on the cell surface of P. gingivalis 11 .
Toll-like receptors are a family of pattern recognition receptors that recognize a plethora of pathogen-associated molecular patterns (PAMPs). To the PAMPs belongs lipopolysaccharide (LPS) from gram-negative bacteria, which is recognized by Toll-like receptors 4 (TLR4) 12 . The importance of TLR4 for periodontal disease is well studied, but much less is known on the role of TLR2. Interestingly, P. gingivalis has the capacity to activate both TLR2 and TLR4 13,14 . Recently, we reported that P. gingivalis stimulates osteoclast formation in vitro and causes inflammation induced bone loss in vivo through activation of TLR2 15 . This observation and the fact that periodontitis induced by P. gingivalis can not be observed in mice with genetic deletion of TLR2 indicates that TLR2 is also important for the pathogenic properties of P. gingivalis in periodontal disease [16][17][18] .
Data from human and mouse studies have evidenced an association between periodontal disease and rheumatoid arthritis (RA) [19][20][21] . The observation that alveolar bone loss in periodontitis patients precede the clinical onset of symptoms of RA 21 , together with the fact that treatment of periodontitis seems to reduce the severity of RA 22,23 indicates a possible cause relationship between the two diseases. Further support for a role of oral infection in RA are studies in mice showing that oral infection with P. gingivalis aggravates arthritic bone erosions in collagen-induced arthritis 22,24 . The pathogenetic mechanisms involved were, at least in part, dependent on Th17 cells through the activation of TLR2 by P. gingivalis 24 . Further supporting an association between periodontal disease and RA is the observation that DNA from P. gingivalis has been detected in serum and synovial fluid from RA patients 25 . The routes used by P. gingvivalis to invade blood vessels in the periodontium and to reach the joints through the circulation are still unknown, but may be attributed to local activation in the periodontal tissues of the kallikrein-kinin system. This hypothesis is supported by the fact that local vascular permeability and bacterial spreading can be enhanced by P. gingivalis through a mechanism that was inhibited by decreasing kinin activity, either by administration of angiotensin converting enzyme (ACE), acting as a kininase enzyme, or by a kinin B2 receptor antagonist. In contrast, increased kinin activity by administration of BK, or the ACE inhibitor captopril, enhanced vascular permeability and bacterial spreading induced by infection with P. gingivalis 26 . Interestingly, the ability of P. gingivalis to disseminate was strain specific and correlated to generation of kinin activity. Thus, local regulation of kinin receptors in gingival fibroblasts could contribute by increasing the response to BK, leading to the generation of vasoactive mediators, such as prostaglandins, and by promoting bacterial spreading and aggravation of RA in periodontitis patients. In the present study, we have investigated the role of TLR2 for the local regulation of kinin receptors and report the novel finding that activation of TLR2 directly increases the expression of functional B 1 and B 2 receptors in human gingival fibroblasts as well as in mouse gingival tissue.

Results
Induction of BDKRB1 and BDKRB2 mRNA expression by P. gingivalis Lps and pam 2 CsK 4 in HGF. Human gingival fibroblasts were isolated from an individual without any clinical signs of gingival inflammation. Exposure of these cells to P. gingivalis LPS (10 μg/ml) for 3-24 hours resulted in time-dependent increased expression of both BDKRB1 (Fig. 1A) and BDKRB2 mRNA (Fig. 1B). The upregulation of BDKRB1 and BDKRB2 mRNA caused by P. gingivalis LPS was concentration dependent, with stimulatory effects seen at and above 100 ng/ml (Fig. 1C,D). Expression of IL6 mRNA has previously been reported to be upregulated by P. gingivalis LPS 27 ; in the present experiments, increased IL6 mRNA was seen at the same concentrations as those stimulating kinin receptor expression (data not shown). BDKRB1 and BDKRB2 mRNA expression was enhanced also by the synthetic TLR2 agonist Pam 2 CSK 4 (50 ng/mL) (Fig. 1E).
In order to evaluate if regulation of kinin receptor expression by P. gingivalis LPS and Pam 2 CSK 4 in gingival fibroblasts was a general phenomenon, we incubated cells isolated from five different individuals with these test substances. In cells from all five individuals, Pam 2 CSK 4 (50 ng/mL) significantly increased both BDKRB1 and BDKRB2 mRNA expression (Fig. 2). P. gingivalis LPS (1 μg/mL) significantly increased both BDKRB1 and BDKRB2 mRNA expression in cells from four of the five patients (Fig. 2).
Induction of Bdkrb1 and Bdkrb2 mRNA expression by P. gingivalis Lps in mouse gingiva. In order to assess if the upregulation of BK receptors by P. gingivalis LPS observed in the fibroblasts cultures could be observed also in vivo, we locally exposed gingival tissue in mice to the bacterial LPS. Injection of P. gingivalis LPS (3 μg) every other day for 14 days in mouse gingiva enhanced the mRNA expression of Bdkrb1 and Bdkrb2 (Fig. 3A,B). Bdkrb1 mRNA was increased by 2.3-fold (Fig. 3A), while Bdkrb2 mRNA was increased by 1.6-fold (Fig. 3B).
Lps from P. gingivalis and pam 2 CsK 4 up-regulate kinin receptor transcripts selectively via TLR2. In order to confirm that up-regulation of kinin receptors by the TLR2 receptor agonists used was a specific effect of TLR2 receptor activation, we knocked down TLR2 by using small interfering RNA designed to silence TLR2 (TLR2-siRNA). To rule out the contribution of TLR4, we also silenced TLR4 using TLR4-siRNA. The mRNA expression levels of TLR2 and TLR4 were decreased by 90%, as compared to cells transfected with a control (scrambled) siRNA (SCR-siRNA; data not shown). Our results showed that knockdown of TLR2 significantly decreased the enhancement of BDKRB1 and BDKRB2 mRNA induced by P. gingivalis LPS, as well as by Pam 2 CSK 4 (Fig. 4A,B). In contrast, knockdown of TLR4 did not significantly affect kinin receptor expression induced by P. gingivalis LPS or by Pam 2 CSK 4 (Fig. 4C,D).
www.nature.com/scientificreports www.nature.com/scientificreports/ TLR2 agonists up-regulate kinin receptors at protein level. As shown in Fig. 5A To analyze the functional relevance of the up-regulation of B1 and B2, we took advantage of the fact that activation of both receptors are linked to increased formation of PGE 2 in gingival fibroblasts 28,29 , as well as in many other cell types. When the fibroblasts were pre-treated with Pam 2 CSK 4 for 6 h (50 ng/mL), the subsequent PGE 2 responses to both BK and DALBK were enhanced (Fig. 5C), indicating that activation of TLR2 results in increased number of functional kinin receptors.

Analysis of the participation of IL-1β and TNF-α in P. gingivalis Lps-induced kinin receptor expression.
In the human gingival fibroblasts, LPS from P. gingivalis induced the expression of IL-1β and TNF-α mRNAs, which were undetectable in control cells not exposed to LPS (data not shown). We, therefore, evaluated if these cytokines could participate in kinin receptor expression induced P. gingivalis LPS and for these purpose made use of specific neutralizing antibodies tested to verify their effectiveness (Supplemental 2). Neither the antibody neutralizing IL-1β (Fig. 6A), nor the one neutralizing TNF-α (Fig. 6C), affected P. gingivalis LPS (1 μg/mL)-induced increase of the mRNA expression of BDKRB1. At variance, although the treatment with . P. gingivalis LPS dose dependently increased mRNA expression of BDKRB1 (C) and BDKRB2 (D) in human gingival fibroblasts after 6 h of treatment with LPS. Pam 2 CSK 4 (50 ng/mL) increased BDKRB1 and BDKRB2 mRNA in human gingival fibroblasts after 6 h of treatment (E). Data were normalized against RPL13A and are expressed as percentage of the means for the controls at 3 h (A) or controls (B-E), which was arbitrarily set to 100%. Values represent means for 3 wells/experimental group and SEM is shown as vertical bar. * and ** indicate significant difference to untreated control cells, P < 0.05 and P < 0.01, respectively. Statistical significance was determined using Student's t test (A, B and E) or one-way analysis of variance (ANOVA), with Levene's homogeneity test and Dunnet's T3 post hoc test (C,D).

Discussion
In the present study, we report that the mRNAs encoding for the B 1 and B 2 kinin receptors are among those genes regulated by LPS from the periodontopathogenic bacterium P. gingivalis, both in vitro in human gingival fibroblasts and in vivo in mouse gingiva. Interestingly, it has been demonstrated that expression of TLR2 mRNA and protein, one of the receptors activated by P. gingivalis is enhanced by activation of B2 kinin receptor, indicating a bidirectional regulation of kinin receptors and TLR2 by their cognate ligands 30 .
In order to escape from the host recognition by the innate immune system and promote its adaptive fitness in the mammalian host, P. gingivalis LPS may elicit different responses when bound to TLR2 or TLR4 13,14 . The heterogeneous responses of P. gingivalis LPS observed in vitro and in vivo may be due to the fact that many preparations are contaminated with lipoproteins or other lipid species 31,32 . Although TLRs are mainly present in inflammatory cells, it has been shown that gingival fibroblasts express a number of proteins belonging to the were upregulated after 6 h of exposure to P. gingivalis LPS (1 μg/mL) or Pam 2 CSK 4 (50 ng/mL) in cells isolated from five individuals. Each bar represents the average of 3 wells/experimental group and SEM is given as vertical bars. *** and *** indicate significant difference to untreated control cells, P < 0.05, P < 0.01 and P < 0.001 respectively. Statistical significance was determined using one-way analysis of variance (ANOVA), with Levene's homogeneity test and Dunnet's T3 post hoc test.
www.nature.com/scientificreports www.nature.com/scientificreports/ TLR family, including TLR2 and TLR4 33 . Such data suggest that resident gingival fibroblasts might participate in the first recognition of pathogens. In line with this observation, we report here that P. gingivalis LPS was able to upregulate B 1 and B 2 receptors, both at the mRNA and at the protein levels. Up-regulation of B 1 and B 2 receptors in an organ culture model of tracheal segments isolated from mice by Salmonella LPS (TLR2 and TLR4 agonist) and by polyinosinic polycytidylic acid (TLR3 agonist) has also been reported 34 . Here, we show that the induction of B 1 and B 2 receptor expression by P. gingivalis LPS is an effect directly mediated by TLR2. One evidence for this is that Pam 2 CSK 4 , a specific TLR2 synthetic agonist, also upregulated B 1 and B 2 receptors in the human gingival fibroblasts. Furthermore, we also demonstrated that the effects elicited by P. gingivalis LPS and Pam 2 CSK 4 were decreased in gingival fibroblasts in which TLR2 expression was robustly decreased by siRNA silencing, but remained unchanged in cells in which TLR4 was likewise siRNA-silenced, although we can not exclude that some remaining TLR4 protein after silencing might have contributed to the response.
It was previously reported that E. coli LPS is capable of regulating the expression of Bdkrb1 in a mouse paw edema model 35 . In this multicellular system, the purposed sequence of events that leads to the up-regulation of B 1 receptor by E. coli involves the release of pro-inflammatory cytokines such as IL-1β and TNF-α, the release of chemoattractant molecules and neutrophil influx. Some years later, it was shown that LPS from P. gingivalis also up-regulates Bdkrb1 in the same model, by a mechanism that also involves neutrophil influx and TNF-α production 4 . Noteworthy, in the present study we show that human gingival fibroblasts are capable of up-regulating both BDKRB1 and BDKRB2 independently of IL-1β and in the case of BDKRB2, partially dependent of TNF-α.
We have previously reported that IL-1β and TNF-α enhance the expression of BDKRB1 and BDKRB2 in human gingival fibroblasts 3 . Since induction of pro-inflammatory cytokines is a well-recognized response to TLR2 activation 36 , we investigated if these cytokines mediated the effects by P. gingivalis LPS and Pam 2 CSK 4 on kinin receptor expression. Using antibodies that specifically neutralize the effects of IL-1β and TNF-α, we show that up-regulation of BDKRB1 occurs independently of the production of both cytokines, whereas BDKRB2 up-regulation is partially dependent on TNF-α production but independent on IL-1β. In the mouse paw model, where inflammatory cells can be recruited to the inflamed site, neutrophil influx and TNF-α production are important events for the regulation of BDKRB1 levels by P. gingivalis LPS 4 . Although TNF-α expressing neutrophils are present in the inflamed gingiva during periodontitis 37 , the up-regulation of BDKRB1 in gingival fibroblasts, independently of IL-1β and TNF-α, may be of importance for the actions of kinins in the periodontium in chronic inflammation. As regards the BDKRB2 up-regulation, it has previously been shown that the up-regulation of this receptor by cardiac myocytes challenged with LPS was partially dependent on TNF-α production 38 , in agreement with our data. Nevertheless, it is interesting to note that the BDKRB2 up-regulation by P. gingivalis LPS was not completely inhibited by TNF-α neutralizing antibody in the gingival fibroblasts, which means that other TNF-α-independent pathways may also be involved in the regulation of the expression of this receptor.
The data presented here may be of clinical relevance, since activation of TLR2 by P. gingivalis is associated with the aggravation of experimental arthritis in mice 24 . In humans, an association between periodontitis and RA has been demonstrated 21,22,24 . One possible mechanism has been suggested to be due to the presence of PAMPs derived from oral bacteria in the diseased joints. Supporting this view, DNA from periodontopathogenic bacteria can be detected in the serum and synovial fluid from patients with RA and psoriatic arthritis 25,39 . The mechanism underlying the invasion of periodontogenic bacteria is still elusive, but one possible route could be local activation of the kallikrein-kinin system from a sequestered infection site to promote vasodilation and facilitate invasion 26 . Our data can be reconciled with this hypothesis, since activation of TLR2 by P. gingivalis LPS not only increased . The expression was analysed using Taqman assays. Data were normalized against Actb and are expressed as percent of the means for the controls, which was arbitrarily set to 100%. Each symbol represents data from one mouse. The horizontal line represents the mean for each experimental group. ** and *** indicate significant difference to untreated mice, P < 0.01 and P < 0.001, respectively. Statistical analysis was determined using Student's unpaired t-test. (2019) 9:2973 | https://doi.org/10.1038/s41598-018-37777-z www.nature.com/scientificreports www.nature.com/scientificreports/ B1 and B2 receptors mRNA, but also increased the capacity of gingival fibroblasts to produce prostaglandin E2, a potent vasodilator agent 40 , in response to the kinins. Kinins themselves are also vasodilatory agents, and can be generated at the inflammatory site during periodontal infection by the action of gingipain, a kinin-producing protease expressed by P. gingivalis 41 . The proposed sequence of events involved in bacterial invasion promoted by P. gingivalis, including a role of TLR2 induced kinin expression in fibroblasts, is outlined in Fig. 7.
In conclusion, in this study we report that P. gingivalis LPS is able to up-regulate kinin receptors in human gingival fibroblasts and mouse gingiva by the activation of TLR2. Moreover, our data reveal a new pathway by which these receptors are up-regulated which is independent on the production of IL-1β and TNF-α in the case of B 1 receptor, and partially dependent on TNF-α production in the case of B 2 receptor. These findings open new horizons for studies investigating mechanisms controlling the expression of B 1 and B 2 receptors in non-inflammatory cells.

Materials. Specified in Supplementary Material 1.
Cell culture. Human gingival fibroblasts were isolated from healthy donors with written, informed consent as previously described 42 . Fibroblasts were from different individuals (males and females between 25-50 years of age (all generally and periodontally healthy) and the cells used in the present study were from passage 5-10. Approval from the Ethical Committee for Human Research at Umeå University was obtained for all the methods described, and all methods were performed in accordance with the relevant guidelines and regulations. The data shown were obtained using cells from one individual but reproduced using cells from another individual with the exception of the experiments used to produce data shown in Fig. 2 which were performed using cells from five different individuals. Twenty-four hours after transfection, the cells were exposed to LPS form P. gingivalis (1 μg/mL) or Pam 2 CSK 4 (50 ng/mL). After 6 h, the expression of BDKRB1 (A,C) and BDKRB2 (B,D) mRNA was analyzed by qPCR using Taqman Assays. Data were normalized against RPL13A and expressed as percent of control which was arbitrarily set to 100%. Data are expressed as means ± SEM (n = 4 wells/experimental group). *, ** and *** indicate significant difference, P < 0.05, P < 0.01 and P < 0.001, respectively. Statistical analysis was determined using two-way analysis of variance (ANOVA), with Levene's homogeneity test and Tukey post hoc test. The difference in P. gingivalis-induced response with and without silencing analyzed by two-way ANOVA was statistically significant (interaction P value in (A) and (B) was P < 0.01). (2019) 9:2973 | https://doi.org/10.1038/s41598-018-37777-z www.nature.com/scientificreports www.nature.com/scientificreports/ In vivo regulation of Bdkrb1 and Bdkrb2 by P. gingivalis LPS. In order to assess the effect of P. gingivalis LPS on the regulation of kinin receptors in vivo, we injected LPS from P. gingivalis (3 μg per injection), or PBS, in the gingiva in the mesial aspect of upper first molar of male 6-weeks old C57Bl/6 mice. The protocol for this experiment was approved by the Ethical Committee on Animal Experimentation at the School of Dentistry Figure 5. TLR2 activation by Pam 2 CSK 4 enhances the number of B1 and B2 binding sites, and the prostaglandin response induced by kinins. Gingival fibroblasts were pre-treated with Pam 2 CSK 4 (50 ng/ml) or PBS (controls) for 24 h (A-C). The cells were then exposed to radiolabelled ligands for 90 minutes for binding analysis, (A,B) or treated for additional 24 h with kinins in order to assess the amount of PGE 2 released (C). The results represent means ± SEM of 4 wells/experimental group. ** and *** indicate significant difference, P < 0.01 and P < 0.001, respectively. Statistical analysis was determined using Student's unpaired t-test (A,B) or determined using two-way analysis of variance (ANOVA), with Levene's homogeneity test and Tukey post hoc test. (C) The difference in PGE 2 release with and without pretreatment with Pam 2 CSK 4 by two-way ANOVA was statistically significant (interaction P value was P < 0.01).
www.nature.com/scientificreports www.nature.com/scientificreports/ in Araraquara -UNESP, Brazil and performed in accordance with the guidelines from the Brazilian College for Animal Experimentation (COBEA). Injections were performed every second day for 14 days, and the animals were sacrificed 6 h after the last injection. The gingival tissue was dissected and the RNA was extracted using RNAqueous-MICRO kit for qPCR analysis.

RNA extraction and cDNA synthesis.
After exposure to the test substances for the time indicated in the graphs or figure legends, total RNA was extracted from the cells using RNAqueous-4PCR kit. The cDNA was synthesized with a first-strand cDNA synthesis kit using oligo(dT) 15 as primers following the manufacturer's instructions.
Quantitative real-time polymerase chain reaction (qPCR). The mRNA expression of human BDKRB1, BDKRB2, TLR2, TLR4 and the mouse genes Bdkrb1 and Bdkrb2 were assessed using previously described primer sequences 3,27 . Amplification was performed in an ABI Prism 7900HT sequence detection system using cDNA as template, specific primers and probes and Taqman Universal Mater Mix kit. To control the amount of cDNA input, ribosomal protein L13A (RPL13A) or β-actin (Actb) were used as controls (housekeeping genes) for human and mouse samples, respectively.

Radioligand binding assays.
After overnight attachment of the fibroblasts, the media were changed and α-MEM with 1% FCS with or without Pam 2 CSK 4 (50 ng/mL) was added. Twenty-four hours later, binding studies were performed following all the standardizations described previously 9 . To assess the amount of binding sites, the cells were incubated in MEM/HEPES/0.1% BSA with [ 3 H]-BK 4 nmol/l or [ 3 H]-des-Arg 10 -Lys-BK 14 nmol/l for 90 min at 4 °C. After extensive washing steps, the cells were detached and the radioactivity analyzed using Figure 6. The role of IL-1β and TNF-α in P. gingivalis LPS mediated up-regulation of BDKRB1 and BDKRB2 mRNA. Gingival fibroblasts were exposed to 1 μg/mL of LPS from P. gingivalis for 6 h in the presence or absence of anti-IL-1β (0.3 μg/mL) (A,B) or anti-TNF-α (1 μg/mL) (C,D) and the expression of BDKRB1 (A,C) and BDKRB2 (B,D) mRNA was analyzed by qPCR using Taqman Assays. Data were normalized against RPL13A and expressed as percent of control which was arbitrarily set to 100%. Data are expressed as means ± SEM (n = 4 wells/experimental group. *** and *** indicate significant difference, P < 0.05, P < 0.01 and P < 0.001, respectively. Statistical analysis was determined using one-way analysis of variance (ANOVA), with Levene's homogeneity test and Tukey post hoc test. prostaglandin e 2 production. The amount of PGE 2 was measured in the supernatant of cells exposed to BK (1 μM) or DALBK (1 μM) for 24 hours by using a commercially available ELISA kit for PGE 2 . In order to analyze the effect of TLR2 activation on kinin receptors expression, cells were pre-treated with or without Pam 2 CSK 4 (50 ng/mL) for 24 h prior to the addition of kinins.
TLR2 and TLR4 knockdown. TLR2 and TLR4 were knocked down in gingival fibroblasts using siRNA as previously described 27 . Briefly, the cells were transfected with 30 nM of scrambled (SCR -Ambion, AM4635), TLR2 (Ambion, ID#111285) or TLR4 siRNA (Ambion, ID#112337) using lipofectamin 2000 in α-MEM with 10% FCS without antibiotics. The knockdown was confirmed by qPCR and more than 90% inhibition of TLR2 and TLR4 mRNA was achieved (data not shown). Twenty-four hours after transfection, the media were changed and the cells were exposed to the test substances; 6 h later RNA was extracted for qPCR analysis.

Participation of IL-1β and TNF-α in up-regulation of kinin receptors induced by P. gingivalis
Lps. After overnight attachment, human gingival fibroblasts were incubated in α-MEM/1% FCS in the presence or absence of P. gingiavlis LPS, with or without antibodies neutralizing human IL-1β or human TNF-α. The IL-1β neutralizing antibodies blocked IL-1β induced enhancement of BDKRB1 mRNA expression and the TNF-α neutralizing antibodies blocked TNF-α induced increase of BDKRB1 mRNA (Supplementary Material 2). statistical analyses. Statistical analysis of multiple treatment groups was performed using analysis of variance (ANOVA), with Levene's homogeneity test, and Dunnet's T3 or Tukey post hoc test. For the experiments with two groups, the unpaired Student's t-test was performed. The data shown in the figures are expressed as means ± standard error of means (SEM) for 3-6 wells per experimental group.

Figure 7.
Proposed role of kinin receptors in gingival fibroblasts for the invasion of P. gingivalis in gingival blood vessels. LPS from P. gingivalis is released from the biofilm on teeth at the inflammatory site and binds to TLR2, composed either by the heterodimer TLR1/TLR2 or TLR6/TLR2 in the cell membrane of human gingival fibroblasts. At the same time, the kinin-releasing protease gingipain expressed by P. gingivalis promotes the generation of kinins at the inflammatory site. Activation of TLR2 leads to the expression of kinin receptor mRNA and protein by gingival fibroblasts. The binding of BK and DALBK to B2 and B1 receptors, respectively, expressed by the fibroblasts leads to the release of PGE 2 . Kinins and PGE2 may act as vasodilator agents, facilitating the penetration of bacteria into the blood vessels and their spreading to other tissues.