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. 2008 Sep 9;105(36):13532-7.
doi: 10.1073/pnas.0803852105. Epub 2008 Sep 2.

Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function

George Hajishengallis  1 , Min Wang, Shuang Liang, Martha Triantafilou, Kathy Triantafilou
Affiliations

Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function

George Hajishengallis et al. Proc Natl Acad Sci U S A. .

Abstract

We report a mechanism of microbial evasion of Toll-like receptor (TLR)-mediated immunity that depends on CXCR4 exploitation. Specifically, the oral/systemic pathogen Porphyromonas gingivalis induces cross-talk between CXCR4 and TLR2 in human monocytes or mouse macrophages and undermines host defense. This is accomplished through its surface fimbriae, which induce CXCR4/TLR2 co-association in lipid rafts and interact with both receptors: Binding to CXCR4 induces cAMP-dependent protein kinase A (PKA) signaling, which in turn inhibits TLR2-mediated proinflammatory and antimicrobial responses to the pathogen. This outcome enables P. gingivalis to resist clearance in vitro and in vivo and thus to promote its adaptive fitness. However, a specific CXCR4 antagonist abrogates this immune evasion mechanism and offers a promising counterstrategy for the control of P. gingivalis periodontal or systemic infections.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CXCR4 associates with TLR2 in Pg-fimbria-activated cells. (A) Human monocytes were pretreated or not with MCD (10 mM) and stimulated with Pg-fimbriae (1 μg/ml, 10 min). FRET between TLR2 (Cy3-labeled) and CXCR4, CD14, or MHC class I (Cy5-labeled) was measured from the increase in donor (Cy3) fluorescence after acceptor (Cy5) photobleaching. (B) MCD effect on TLR2 or CXCR4 surface expression using FACS. (C) Association of CXCR4 with GM1 (lipid raft marker) in Pg-fimbria-activated monocytes, determined by FRET. (D) Confocal colocalization of FITC-P. gingivalis with both CXCR4 and TLR2 in human monocytes (Upper) or mouse macrophages (Lower). Data are means ± SD (n = 3). Asterisks show significant (P < 0.01) differences vs. medium-only control. Black circles indicate significant (P < 0.01) reversal of FRET increase.
Fig. 2.
Fig. 2.
CXCR4 regulates human monocyte activation in response to Pg-fimbriae. Monocytes were stimulated with Pg-fimbriae with or without pretreatment with anti-CXCR4 mAb or isotype control (5 μg/ml). After 90 min, cellular extracts were analyzed for NF-κB p65 activation (A). After 16 h, culture supernatants were assayed for TNF-α (B) or IL-10 (C). Data are means ± SD (n = 3) from one of three independent sets of experiments yielding similar results. Asterisks show significant (P < 0.01) differences vs. IgG2a isotype and medium-only controls.
Fig. 3.
Fig. 3.
Pg-fimbriae bind to CXCR4. (A) Empty vector- or CXCR4-transfected CHO cells were pretreated with AMD3100 (1 μg/ml), anti-CXCR4 mAb, IgG2a isotype control, irrelevant mAb (5 μg/ml), or 100-fold excess unlabeled fimbriae, and then incubated with biotinylated fimbriae (1 μg/ml). (B) Similar experiment, without inhibitors, using increasing concentrations of ligand. Binding was measured as cell-associated fluorescence after staining with streptavidin (SA)-FITC. Data are means ± SD (n = 3) from typical experiments performed three (A) or two (B) times yielding similar findings. In A, the asterisk indicates significant increase in binding (P < 0.01 vs. vector control) and black circles denote significant (P < 0.01) inhibition of binding.
Fig. 4.
Fig. 4.
CXCR4 inhibits TLR2-induced NF-κB activation in response to Pg-fimbriae. (A) CHO cells were transfected with human CD14 and TLR2 with or without CXCR4. Both groups as well as empty vector-transfectants were cotransfected with NF-κB reporter system. After 48 h, the cells were stimulated for 6 h with Pg-fimbriae (1 μg/ml). NF-κB activation is reported as relative luciferase activity (RLA). (B) CHO-CD14/TLR2/CXCR4 cells assayed as in A, except that CXCR4 was blocked by AMD3100 (1 μg/ml) or anti-CXCR4 (5 μg/ml). (C) NF-κB activation in CHO-CD14/TLR2/CXCR4 cells in response to increasing concentrations of Pg-fimbriae in the presence of anti-CXCR4 or IgG2a isotype control. Results are means ± SD (n = 3) from one set of experiments that was repeated yielding similar findings. Asterisks show significant differences in NF-κB activation (A and B, P < 0.01; C, P < 0.05). The controls against which comparisons were made were CHO-CD14/TLR2 cells (A), medium only (B), or IgG2a control (C).
Fig. 5.
Fig. 5.
Inhibitors of cAMP and PKA reverse CXCR4-mediated suppression of NF-κB activation. CHO cells were cotransfected with human CD14, TLR2, and CXCR4, and with NF-κB reporter system. After 48 h, the transfectants were pretreated as indicated and stimulated for 6 h with Pg-fimbriae (1 μg/ml) (A) or whole cells of P. gingivalis (moi = 10:1) (B). The concentrations used were: 1 μg/ml AMD3100, 200 μM SQ22536, 10 μM H89, 1 μM chelerythrin, 1 μM PKI 6–22 (peptide inhibitor of PKA), 1 μM KT5823 (peptide inhibitor of PKG; control). NF-κB activation is reported as RLA and the horizontal lines indicate the level of NF-κB activation in Pg-fimbria-stimulated CHO cells transfected with CD14 and TLR2 only. Data are means ± SD (n=3) of typical experiments performed three (A) or two (B) times yielding similar results. Asterisks show significant (P < 0.01) up-regulation of NF-κB activation vs. no-inhibitor control. (C) Summarizing model of the data. Unlike CD14, which facilitates TLR2 activation by P. gingivalis, CXCR4 suppresses TLR2-mediated NF-κB activation by inducing inhibitory cAMP-dependent PKA signaling.
Fig. 6.
Fig. 6.
CXCR4 blockade inhibits P. gingivalis survival in vitro and in vivo in a NO-dependent way. (A–D) Mouse macrophages were treated with the indicated inhibitors or controls at these concentrations: 1 μg/ml AMD3100, 15 μg/ml anti-CXCR4 mAb or isotype control, 200 μM SQ22536, 10 μM H89, 1 μM chelerythrin, 1 μM PKI 6–22, and 1 μM KT5823. The cells were then infected with P. gingivalis (moi = 10:1). After 24 h, production of NO2− was assayed by the Griess reaction (A and C), and viable CFU of internalized bacteria were determined by using an intracellular survival assay (B and D). (A Inset) NO2− production in P. gingivalis-stimulated wild-type or TLR2−/− macrophages. (E and F) BALB/c mice i.p. pretreated or not with AMD3100 (25 μg in 0.1 ml of PBS) with or without L-NAME or D-NAME (0.1 ml of 12.5 mM solution), as indicated. After 1 h, the mice were i.p. infected with P. gingivalis (5 × 107 CFU). The administration of pretreating agents was repeated 8 h postinfection. Peritoneal fluid was collected 20 h postinfection and used to determine viable P. gingivalis CFU (E) and NO2− production (F). Data are from one of two independent sets of experiments yielding similar findings, and are presented as means ± SD (A–D, n = 3; F, n = 5) or are shown for each mouse with horizontal lines indicating mean values (E). The asterisks show significant (P < 0.01) differences vs. medium-only treatments (A–D), vs. wild-type macrophages (A Inset), or vs. PBS-treated groups (E and F).
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