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, 9 (1), 2973

Activation of Toll-like Receptor 2 Induces B 1 and B 2 Kinin Receptors in Human Gingival Fibroblasts and in Mouse Gingiva

Affiliations

Activation of Toll-like Receptor 2 Induces B 1 and B 2 Kinin Receptors in Human Gingival Fibroblasts and in Mouse Gingiva

Pedro P C Souza et al. Sci Rep.

Abstract

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
P. gingivalis LPS and the TLR2 agonist Pam2CSK4 increase the expression of BDKRB1 and BDKRB2 mRNA in human gingival fibroblasts. Time-course of the expression of BDKRB1 and BDKRB2 in human gingival fibroblasts cultured in the presence or absence of 10 μg/mL of P. gingivalis LPS (A,B). 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. Pam2CSK4 (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 (BE), 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).
Figure 2
Figure 2
P. gingivalis LPS and the TLR2 agonist Pam2CSK4 increase the expression of B1 and B2 kinin transcripts in human gingival fibroblasts from different individuals. BDKRB1 (A) and BDKRB2 (B) were up-regulated after 6 h of exposure to P. gingivalis LPS (1 μg/mL) or Pam2CSK4 (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.
Figure 3
Figure 3
P. gingivalis LPS increases the expression of Bdkrb1 and Bdkrb2 in mouse gingiva. Injection of LPS from P. gingivalis (3 μg) every other day for 14 days increases the expression of Bdkrb1 (A) and Bdkrb2 (B) in mouse gingiva in comparison with injection of vehicle (Control). 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.
Figure 4
Figure 4
The up-regulation of BDKRB1 and BDKRB2 mRNA by LPS from P. gingivalis is mediated by TLR2. Gingival fibroblasts were transfected with a scrambled siRNA or siRNA targeting TLR2 (A,B) or TLR4 (C,D). Twenty-four hours after transfection, the cells were exposed to LPS form P. gingivalis (1 μg/mL) or Pam2CSK4 (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).
Figure 5
Figure 5
TLR2 activation by Pam2CSK4 enhances the number of B1 and B2 binding sites, and the prostaglandin response induced by kinins. Gingival fibroblasts were pre-treated with Pam2CSK4 (50 ng/ml) or PBS (controls) for 24 h (AC). 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 PGE2 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 PGE2 release with and without pretreatment with Pam2CSK4 by two-way ANOVA was statistically significant (interaction P value was P < 0.01).
Figure 6
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.
Figure 7
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 PGE2. Kinins and PGE2 may act as vasodilator agents, facilitating the penetration of bacteria into the blood vessels and their spreading to other tissues.

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