Induction of distinct TLR2-mediated proinflammatory and proadhesive signaling pathways in response to Porphyromonas gingivalis fimbriae

Abstract

The oral pathogen Porphyromonas gingivalis, as well as its purified fimbriae, are known to activate TLR2 and induce proinflammatory and proadhesive effects. The TLR2 proinflammatory pathway induces NF-kappaB-dependent inflammatory cytokines, whereas the TLR2 proadhesive pathway is characterized by inside-out signaling that transactivates beta(2) integrin adhesive activities. In this article, using dominant-negative or pharmacological approaches, we show that the two pathways bifurcate and proceed independently downstream of TLR2. Whereas the proinflammatory pathway is dependent on the adaptor molecules Toll/IL-1 receptor domain-containing adaptor protein (also known as Mal) and MyD88, the proadhesive pathway is Toll/IL-1 receptor domain-containing adaptor protein/MyD88-independent and proceeds through PI3K-mediated signaling. Although the Ser/Thr kinase Akt is a major downstream target of PI3K and was activated by P. gingivalis fimbriae in a TLR2- and PI3K-dependent way, Akt was shown not to play a role in the proadhesive pathway. In contrast, another PI3K downstream target, cytohesin-1, was shown to mediate P. gingivalis fimbria-induced activation of beta(2) integrin for ICAM-1 binding. Therefore, P. gingivalis fimbriae activate two distinct TLR2 pathways mediating proinflammatory or proadhesive effects. The delineation of these signaling pathways may provide appropriate targets for selectively inhibiting or enhancing specific activities, depending on whether they undermine or promote the host defense.

Figures

Figure 1. P. gingivalis fimbria-induced activation of human CR3 is MyD88-independent

Human monocytic THP-1/CD14 cells were transfected with a dominant negative mutant of MyD88 (MyD88-DN), TLR2-DN, or vector control, at the indicated μg of plasmid DNA per 2×105 cells (A), or were pretreated for 2h with MyD88 inhibitory peptide or inactive control (100 μM) (B). The cells were stimulated with P. gingivalis fimbriae (1 μg/ml) and assayed for induction of the CR3 activation-specific CBRM1/5 epitope (after staining with FITC-labeled CBRM1/5 mAb) or for NF-κB-dependent transcription of a luciferase reporter gene. CBRM1/5 induction is reported in relative fluorescent units (RFU) and NF-κB activation in relative luciferase activity (RLA). The discontinuous horizontal lines indicate CBRM1/5 epitope induction or NF-κB activation in resting cells, which were < 8% of the values of fimbria-stimulated cells. Results are presented as means ± SD (n = 3) from one of two sets of experiments yielding consistent results. The asterisks indicate statistically significant (p < 0.05) differences compared to control treatments.

Figure 2. Role of MyD88 in P. gingivalis fimbria-induced proadhesive or proinflammatory activities in mouse macrophages

Mouse macrophages (wild-type or deficient in MyD88 or TLR2) were activated with P. gingivalis fimbriae (1 μg/ml) and assayed for CR3-dependent binding of sICAM-1 (A, B) or induction of TNF-α release (C). In B, wild-type macrophages were pretreated for 30 min with a CR3 blocking (anti-CD11b) mAb or isotype control. The agonists, E. coli LPS (0.2 μg/ml), PMA (0.1 μg/ml), and Poly(I:C) (50 μg/ml), were used as controls in A and/or C, as indicated. Data are presented as means ± SD (n = 3) from one of three (A) or two (B, C) independent experiments that yielded consistent results. Asterisks indicate statistically significant (p < 0.05) differences compared to wild-type macrophages (A, C) or compared to no-antibody treatment (B).

Figure 3. TIRAP-dependent and –independent proinflammatory and proadhesive activities of P. gingivalis fimbriae

Human THP-1/CD14 cells were transfected with dominant negative (DN) point mutants of human TIRAP (TIRAP-DN) or TRAM (TRAM-DN; negative control), or a TIR domain-deficient mutant of TLR2 that acts as a dominant negative inhibitor (TLR2-DN; positive control), at the indicated μg amounts of plasmid DNA per 2×105 cells. The cells were stimulated with P. gingivalis fimbriae (1 μg/ml) and assayed for induction of the CR3 activation-specific CBRM1/5 epitope (after staining with FITC-labeled CBRM1/5 mAb) or for NF-κB-dependent transcription of a luciferase reporter gene. CBRM1/5 induction is reported in relative fluorescent units (RFU) and NF-κB activation in relative luciferase activity (RLA). The discontinuous horizontal lines indicate CBRM1/5 epitope induction or NF-κB activation in resting cells, which were < 12 % of the values of fimbria-stimulated cells. Data are means ± SD (n = 3) from one of two experiments that yielded consistent results. The asterisks indicate statistically significant (p < 0.05) differences compared to empty vector control treatments.

Figure 4. Role of TIRAP in P. gingivalis fimbria-induced proadhesive or proinflammatory activities in mouse macrophages

Mouse macrophages were pretreated for 1h with 10 μM of a cell-permeable TIRAP inhibitory peptide or inactive control. The cells were subsequently left unstimulated or were stimulated with P. gingivalis fimbriae (1 μg/ml) and assayed for sICAM-1 binding or induction of IL-6 release. Data are means ± SD (n = 3) from one of two independent experiments that yielded consistent findings. Asterisks indicate statistically significant (p < 0.05) differences compared to medium-treated (no peptide) controls.

Figure 5. Akt is activated by P. gingivalis fimbriae in a TLR2/PI3K-dependent way but is not involved in CR3 activation

THP-1/CD14 cells were transfected or not with dominant negative inhibitors of TLR2 (TLR2-DN) [A and B] or Akt (Akt-DN) [B] at the indicated μg of plasmid DNA per 2×105 cells. In A, untransfected THP-1/CD14 cells were pretreated with medium only, LY294002 (PI3K inhibitor) or the inactive analog LY30351 (both at 20 μM). Cells were then activated with P. gingivalis fimbriae and assayed for total and phosphorylated Akt [A], or for induction of the CBRM1/5 epitope, measured in relative fluorescence units (RFU) [B]. The discontinuous horizontal line in B indicates CBRM1/5 epitope induction in resting cells which was < 10% of the activity of fimbria-stimulated cells. Data are means ± SD (n = 3), from one of three (A) or two (B) independent experiments yielding consistent results. Asterisks indicate statistically significant (p < 0.05) differences compared to corresponding control treatments.

Figure 6. Cytohesin-1 is involved in P. gingivalis fimbria-induced proadhesive but not proinflammatory activities

THP-1/CD14 cells were treated with antisense S-oligos (AS) to cytohesin-1 mRNA or with control sense S-oligos (S), at the indicated concentrations (μM). Following stimulation with P. gingivalis fimbriae (1 μg/ml), the cells were assayed for induction of the CBRM1/5 epitope, binding of sICAM-1, or for NF-κB-dependent transcription of a luciferase reporter gene. CBRM1/5 induction and sICAM-1 binding are reported in relative fluorescent units (RFU) and NF-κB activation in relative luciferase activity (RLA). Unstimulated cells displayed < 9 % of the activities of fimbria-stimulated cells in all three assays. Results are means ± SD (n = 3) from one of two independent experiments that yielded consistent findings. Asterisks indicate statistically significant (p < 0.05) inhibition of activities compared to cells that were not treated with anti-sense S-oligos.

Figure 7. P. gingivalis-activated TLR2 induces distinct proadhesive and proinflammatory signaling pathways

The model is based on the current findings and our previously published studies (, –21). P. gingivalis fimbriae interact with the CD14/TLR2 complex leading to TLR2 activation. Activated TLR2 induces TIRAP/MyD88-dependent signaling for NF-κB activation and induction of proinflammatory cytokines (proinflammatory pathway), and TIRAP/MyD88-independent signaling for transactivation of the CR3 adhesive activity (proadhesive pathway). The latter pathway proceeds via Rac1, PI3K and cytohesin-1 (Cyt-1). Cytohesin-1 is likely to function immediately downstream of PI3K, since it was previously shown to use PI3K-generated PIP3 for membrane docking and interaction with the CD18 cytoplasmic tail (48).