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. 2013 Aug;28(4):239-49.
doi: 10.1111/omi.12021. Epub 2013 Jan 21.

Mechanism and implications of CXCR4-mediated integrin activation by Porphyromonas gingivalis

G Hajishengallis  1 , M L McIntosh, S-I Nishiyama, F Yoshimura
Affiliations

Mechanism and implications of CXCR4-mediated integrin activation by Porphyromonas gingivalis

G Hajishengallis et al. Mol Oral Microbiol. 2013 Aug.

Abstract

In monocytes and macrophages, the interaction of Porphyromonas gingivalis with Toll-like receptor 2 (TLR2) leads to the activation of a MyD88-dependent antimicrobial pathway and a phosphatidylinositol-3 kinase (PI3K) -dependent pro-adhesive pathway, which activates the β2 -integrin complement receptor 3 (CR3). By means of its fimbriae, P. gingivalis binds CXC-chemokine receptor 4 (CXCR4) and induces crosstalk with TLR2 that inhibits the MyD88-dependent antimicrobial pathway. In this paper, we investigated the impact of the P. gingivalis-CXCR4 interaction on the pro-adhesive pathway. Using human monocytes, mouse macrophages, or receptor-transfected cell lines, we showed that the binding of P. gingivalis fimbriae to CXCR4 induces CR3 activation via PI3K, albeit in a TLR2-independent manner. An isogenic strain of P. gingivalis expressing mutant fimbriae that do not interact with CXCR4 failed to efficiently activate CR3, leading to enhanced susceptibility to killing in vivo compared with the wild-type organism. This in vivo observation is consistent with previous findings that activated CR3 mediates safe entry of P. gingivalis into macrophages. Taken together with our previous work, these results indicate that the interaction of P. gingivalis with CXCR4 leads to inhibition of antimicrobial responses and enhancement of pro-adhesive responses, thereby maximizing its adaptive fitness in the mammalian host.

Keywords: CXCR4; Porphyromonas gingivalis; Toll-like receptor 2; immune evasion; integrin; periodontitis.

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Figures

Figure 1
Figure 1. CXCR4 is involved in P. gingivalis fimbria-induced CR3 activation
(A) Human monocytes were stimulated or not with fimbriae (1μg/ml) for the indicated times, with or without AMD3100 (1μg/ml), and assayed for CBRM1/5 epitope induction using FITC-labeled CBRM1/5 mAb and flow cytometry. FITC-labeled IgG1 control was used to assess background fluorescence (“unstained” group). CBRM1/5 induction is reported in mean fluorescence intensity (MFI) values. (B) Mouse peritoneal macrophages were activated with fimbriae (1 μg/ml), with or without AMD3100 (1 μg/ml) or anti-CXCR4 (5 μg/ml), and assayed for CR3-dependent binding of FITC-labeled sICAM-1 at 30 min following activation. CR3 dependence was confirmed by including groups treated with anti-CR3 (anti-CD11b) mAb or isotype control. sICAM-1 binding is reported in relative fluorescent units (RFU). Data are means ± SD (n = 3). *, statistically significant (P < 0.01) inhibition of CBRM1/5 epitope induction (A) or of sICAM-1 binding (B).
Figure 2
Figure 2. CR3-dependent binding activities of CHO-CR3 cells transfected with TLR2 and/or CXCR4
CHO-CR3 cells, transiently transfected with the indicated receptors or empty vector control, were activated with P. gingivalis fimbriae (1 μg/ml) (A) or whole cells of P. gingivalis (MOI = 10:1) (B) and assayed for CR3-dependent binding of FITC-labeled sICAM-1 at 30 min following activation. CR3 dependence was confirmed by including groups treated with an anti-CR3 (anti-CD11b) mAb or isotype control. sICAM-1 binding is reported in relative fluorescent units (RFU). Data are means ± SD (n = 3). *, statistically significant (P < 0.01) differences between the indicated groups.
Figure 3
Figure 3. CXCR4-dependent activation of CR3 is mediated by PI3K
(A) CHO-CR3 cells, transiently transfected with CXCR4 or empty vector control, were stimulated with P. gingivalis fimbriae (1 μg/ml) and assayed for binding FITC-labeled sICAM-1 at 30 min following activation. Prior to stimulation, the cells were pretreated for 30 min with AMD3100 (1 μg/ml), wortmannin (WTM; 50 nM), LY294002 (20 μM), LY30351 (20 μM), H89 (5 μM), PKI 6-22 (1 μM), or GF109203X (10 μM). (B) Similar experiment in untransfected CHO-CR3 cells which were stimulated with PMA (0.1 μg/ml) or medium-only control. sICAM-1 binding is reported in relative fluorescent units (RFU). Data are means ± SD (n = 3). *, statistically significant (P < 0.01) inhibition of of sICAM-1 binding.
Figure 4
Figure 4. DAP fimbriae bind pre-activated CR3 but do not efficiently activate CR3
(A) CHO-CR3 cells were preactivated with PMA (0.1 μg/ml) and assayed for binding wild-type (WT) or DAP fimbriae (both at 1 μg/ml) after 30-min incubation, in the absence or presence of anti-CD11b mAb or IgG1 isotype control. (B) Human monocytes were stimulated with WT or DAP fimbriae (both at 1 μg/ml) with or without AMD3100 (1 μg/ml) and assayed for CBRM1/5 epitope induction at 30 min following activation. CBRM1/5 induction is reported in mean fluorescence intensity (MFI) values. The horizontal dashed lines indicate background binding (6943 ± 892 RFU) to empty vector-transfected cells (A) or baseline CBRM1/5 induction (8.8 ± 1.7 MFI) in unstimulated cells (B). (C) CHO-CR3 cells, transiently transfected with CXCR4 or empty vector control, were stimulated with WT or DAP fimbriae (both at 1 μg/ml) with or without AMD3100 (1 μg/ml) and after 30 min were assayed for binding of FITC-labeled sICAM-1. Data are means ± SD (n = 3). *, statistically significant (P < 0.01) differences between the indicated groups. NS, not significant.
Figure 5
Figure 5. P. gingivalis expressing DAP fimbriae does not exploit CXCR4 or CR3
BALB/cByJ mice were i.p. pretreated with AMD3100 (25 μg in 0.1 ml PBS), XVA143 (15 μg in 0.1 ml PBS), both AMD3100 and XVA143, or PBS alone. After 1h, the mice were i.p. infected with 5×107 CFU P. gingivalis 33277 or OZ5001C (FimCDE-deficient isogenic mutant). Peritoneal lavage was performed 24h postinfection. Serial 10-fold dilutions of peritoneal fluid were plated for anaerobic growth and enumeration of recovered CFU. Horizontal lines show mean CFU counts. Asterisks indicate significant (P < 0.01) differences in P. gingivalis peritoneal CFU between PBS-treated mice and mice treated with receptor antagonists. The arrow sign shows significant (P < 0.01) difference between dual and single antagonist treatments. The difference between 33277 and OZ5001C CFU in PBS-pretreated mice is statistically significant (p < 0.01).
Figure 6
Figure 6. Exploitation of CXCR4 by P. gingivalis
In macrophages, P. gingivalis interacts with CD14 and the TLR2/TLR1 signaling complex resulting in inside-out signaling for activating and binding CR3, which leads to a relatively ‘safe’ uptake of these organisms by macrophages (Hajishengallis et al., 2007; Hajishengallis et al., 2006; Wang et al., 2007). The signaling pathway that activates the high-affinity state of CR3 is mediated by Rac1, PI3K and cytohesin 1 (Cyt1) (Hajishengallis et al., 2009; Harokopakis et al., 2006; Harokopakis & Hajishengallis, 2005). P. gingivalis-activated TLR2/TLR1 also induces a MyD88-dependnet pathway that can potentially promote the killing of this bacterium (Hajishengallis et al., 2009; Hajishengallis et al., 2008). However, by means of its fimbriae, P. gingivalis instigates a crosstalk between CXCR4 and TLR2 which interferes with this antimicrobial mechanism(Hajishengallis et al., 2008). In this study, P. gingivalis was shown to also utilize CXCR4 to induce PI3K-dependent activation of CR3, independently of TLR2, which further contributes to its capacity to evade killing. CXCR4 exploitation requires fully functional fimbriae, i.e., containing both the FimA and FimCDE components, which can directly bind CXCR4 (Pierce et al., 2009).
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