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

Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function George Hajishengallis*†‡, Min Wang*, Shuang Liang*, Martha Triantafilou§, and Kathy Triantafilou§ *Division of Oral Health and Systemic Disease/Department of Periodontics and †Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292; and §Infection and Immunity Group, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom Edited by Bruce Alan Beutler, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board July 2, 2008 (received for review April 23, 2008)

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 coassociation 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. bacterial pathogenesis 兩 immune evasion 兩 macrophages 兩 P. gingivalis 兩 protein kinase A

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icrobial infection is detected by pattern-recognition receptors, among which Toll-like receptors (TLRs) play a central role in inducing innate immune responses for pathogen control (1). TLRs do not function in isolation but cooperate with other receptors in multireceptor complexes within membrane lipid rafts (2–4). The formation of TLR-containing receptor clusters may serve to generate a combinatorial repertoire for discriminating among the abundant and diverse microbial molecules and thereby to tailor the host response. However, it is conceivable that pathogens may exploit the propensity of TLRs for cooperation with heterotypic receptors by instigating the recruitment of receptors that could deregulate effective innate immunity. In this article, we present evidence that Porphyromonas gingivalis effectively uses this immune evasion strategy. P. gingivalis is a predominant pathogen associated with human periodontitis, an infection-driven chronic inflammatory disease of the oral cavity (5). This Gram-negative anaerobic organism is moreover implicated in systemic conditions such as atherosclerosis (6) or aspiration pneumonia (7). The pathogenic potential of P. gingivalis is attributed to several virulence factors (e.g., fimbriae and cysteine proteinases), which enable the pathogen to colonize or invade host tissues and secure critical nutrients (8). However, a pathogen’s ability to find a niche and establish chronic infection requires more than possessing appropriate adhesins or other factors for nutrient procurement. To persist in a hostile host environment, pathogens should be able to evade or subvert the host immune system aiming to control or eliminate them. Successful pathogenic organisms that disable host defenses target preferentially innate immunity (9). This may also undermine the overall host defense, given the instructive role of innate immunity in the adaptive immune response (1). The fimbriae of P. gingivalis, which comprise polymerized fimbrillin (FimA) and accessory proteins (FimCDE) encoded by genes of the fimbrial operon (10), are traditionally recognized as a major colonization factor (8). In this article, we show that the fimbriae of P. gingivalis (henceforth referred to as Pg-fimbriae) contribute to its virulence also through immune subversion of TLR signaling. By 13532–13537 兩 PNAS 兩 September 9, 2008 兩 vol. 105 兩 no. 36

virtue of their length [up to 3 ␮m (8)], Pg-fimbriae may be the first P. gingivalis molecule to interact with innate immune cells and initiate intracellular signaling. The initial recognition event involves binding of Pg-fimbriae to CD14, which serves as a coreceptor that facilitates TLR2 signaling (3, 11). The outcome of TLR2 activation in response to distinct microbial molecules may be influenced by differential TLR2 association with accessory receptors, as previously shown for TLR4 (12). Here, we have identified CXCchemokine receptor 4 (CXCR4) as a TLR2-associated receptor interacting with Pg-fimbriae and examined a possible cross-talk between the two receptors. Strikingly, unlike CD14, which facilitates TLR2 activation by P. gingivalis (3), CXCR4 appeared to limit TLR2 activation in human monocytes or mouse macrophages. Specifically, we found that Pg-fimbriae induce CXCR4-mediated activation of cAMPdependent protein kinase A (PKA), which in turn inhibits TLR2induced NF-␬B activation in response to P. gingivalis. CXCR4 may thus serve a homeostatic role to prevent excessive TLR2-induced inflammation or, alternatively, CXCR4 may be exploited by P. gingivalis for suppressing TLR2-mediated innate immunity. However, we additionally found that the interaction of P. gingivalis with CXCR4 impairs antimicrobial host defense and promotes the survival of the pathogen in vitro and in vivo. Therefore, P. gingivalis appears to exploit its interaction with CXCR4 as a mechanism of immune evasion. Results Pg-Fimbriae Induce TLR2/CXCR4 Co-Association. Using FRET, we pre-

viously showed that TLR2 is recruited to membrane lipid rafts and associates with CD14 in Pg-fimbria-activated monocytes but not when the rafts are disrupted by cholesterol depletion using methyl␤-cyclodextrin (MCD) (3, 13). Using the same technique, we have now identified CXCR4 as a potential TLR2 coreceptor, in line with earlier observations by some of the coauthors that this receptor is a component of pattern-recognition receptor complexes (14). Specifically, significant energy transfer was detected between Cy3labeled TLR2 (donor) and Cy5-labeled CXCR4 (acceptor) in stimulated but not in resting monocytes (Fig. 1A), indicating that Pg-fimbriae induce TLR2/CXCR4 co-association. As expected, Pg-fimbriae induced TLR2 association with CD14 (positive control) but not with MHC class I (negative control) (Fig. 1 A). Author contributions: G.H., M.T., and K.T. designed research; G.H., M.W., S.L., M.T., and K.T. performed research; G.H., M.W., S.L., M.T., and K.T. analyzed data; and G.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. B.A.B. is a guest editor invited by the Editorial Board. Freely available online through the PNAS open access option. ‡To

whom correspondence should be addressed at: University of Louisville, 501 South Preston Street, Louisville, KY 40292. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/cgi/content/full/ 0803852105/DCSupplemental. © 2008 by The National Academy of Sciences of the USA

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However, treatment of monocytes with MCD before stimulation abrogated the energy transfer between TLR2 and CXCR4 (Fig. 1 A), suggesting that their co-association takes place in lipid rafts. To rule out that MCD causes loss or shedding of TLR2 or CXCR4, we examined their expression in MCD-treated or untreated cells. Indeed, flow cytometry revealed that MCD did not alter the expression of TLR2 or CXCR4 (Fig. 1B). Additional support for lipid raft association was obtained by demonstrating that CXCR4 associates with an established lipid raft marker (GM1 ganglioside) upon cell activation with Pg-fimbriae (Fig. 1C). Consistent with the notion that Pg-fimbriae induce TLR2/CXCR4 co-association, P. gingivalis was found to colocalize with both CXCR4 and TLR2 in human monocytes or mouse macrophages (Fig. 1D). We next investigated the functional significance of TLR2/CXCR4 coassociation in response to Pg-fimbriae. Pg-Fimbriae Interact with CXCR4 and Suppress TLR2-Induced Cell Activation. The ability of Pg-fimbriae to activate monocytes depends

almost exclusively on TLR2 (3). We tested the hypothesis that CXCR4 acts as a TLR2 coreceptor in Pg-fimbria-induced cell activation. Specifically, we speculated that a blocking mAb to CXCR4 would suppress Pg-fimbria-induced cell activation. Surprisingly, Pg-fimbriae induced significantly stronger NF-␬B activation (Fig. 2A) and TNF-␣ production (Fig. 2B) in anti-CXCR4pretreated than in isotype-control-pretreated cells. Strikingly, the immunosuppressive cytokine IL-10 was conversely affected; i.e., it was down-regulated (Fig. 2C). These data indicate that CXCR4 regulates Pg-fimbria-induced cell activation and imply that Pgfimbriae interact directly with CXCR4.

To determine that Pg-fimbriae can indeed bind CXCR4, we used CHO-K1 cells that do not normally express CXCR4 (15) and, moreover, interact poorly with Pg-fimbriae (13). We thus transfected CHO-K1 cells with human CXCR4 and examined the binding of biotinylated Pg-fimbriae probed with streptavidinFITC. Although Pg-fimbriae displayed poor binding to emptyvector-transfected CHO-K1 cells, their binding was increased ⬎3-fold in CXCR4-transfected CHO-K1 cells (henceforth designated CHO-CXCR4 cells) (Fig. 3A). The interaction of fimbriae with CHO-CXCR4 cells involved specific binding to CXCR4 as shown by potent blocking effects of a specific antagonist [AMD3100 (16)] and of anti-CXCR4 mAb, whereas isotype control or irrelevant mAb had no effect (Fig. 3A). The binding specificity was additionally confirmed by the finding that excess unlabeled Pg-fimbriae inhibited the binding of labeled Pg-fimbriae to CHO-CXCR4 cells (Fig. 3A). Incubation of increasing concentrations of ligand with CHO-CXCR4 or control CHO cells showed that Pg-fimbriae bind the former cells in a saturable manner (Fig. 3B). In conclusion, the data from Figs. 2 and 3 are consistent with the intriguing notion that Pg-fimbriae interact with CXCR4 and induce signaling that inhibits NF-␬B activation and TNF-␣ induction on the one hand but promotes IL-10 production on the other. To provide direct evidence that Pg-fimbriae induce CXCR4/ TLR2 cross-talk that suppresses TLR2-mediated NF-␬B activation, we performed the following experiment. CHO-K1 cells were transfected with human CD14 and TLR2 to render them responsive to Pg-fimbriae in terms of NF-␬B activation [CHO-K1 cells express endogenous TLR1 and TLR6, either of which can be used by 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 ␮g/ml). After 90 min, cellular extracts were analyzed for NF-␬B p65 activation (A). After 16 h, culture supernatants were assayed for TNF-␣ (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.

Hajishengallis et al.

PNAS 兩 September 9, 2008 兩 vol. 105 兩 no. 36 兩 13533

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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 ␮g/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-fimbriaactivated 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. 3. Pg-fimbriae bind to CXCR4. (A) Empty vector- or CXCR4-transfected CHO cells were pretreated with AMD3100 (1 ␮g/ ml), anti-CXCR4 mAb, IgG2a isotype control, irrelevant mAb (5 ␮g/ml), or 100-fold excess unlabeled fimbriae, and then incubated with biotinylated fimbriae (1 ␮g/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.

Pg-fimbriae for cooperative TLR2 signaling induction (3)]. A second group was additionally cotransfected with human CXCR4. Both CHO-CD14/TLR2 and CHO-CD14/TLR2/CXCR4 cells, as well as empty vector-transfected cells, were subsequently compared for their potential to activate NF-␬B-dependent transcription in response to Pg-fimbriae. As expected, Pg-fimbriae could readily activate NF-␬B in CHO-CD14/TLR2 cells (Fig. 4A). However, stimulation of CHO-CD14/TLR2/CXCR4 cells with Pg-fimbriae resulted in significant inhibition (59%) of NF-␬B activation (Fig. 4A), which was reversed upon CXCR4 blockade with AMD3100 or anti-CXCR4 mAb (Fig. 4B). Interestingly, similar experiments performed in human embryonic kidney-293 cells revealed that CXCR4 inhibits TLR2-induced cell activation by Pg-fimbriae regardless of whether TLR1 or TLR6 is cotransfected to serve as a signaling partner [supporting information (SI) Fig. S1]. However, CXCR4 does not inhibit TLR2 signaling in a nonspecific way, because it did not affect TLR2/TLR1 or TLR2/TLR6 signaling by the lipopeptides Pam3Cys and MALP-2, respectively (Fig. S1). The up-regulatory effect of CXCR4 blockade on NF-␬B activation by Pg fimbriae was observed over a wide concentration range of the agonist (Fig. 4C). Therefore, these data strongly suggest that CXCR4 inhibits TLR2-dependent NF-␬B activation in response to Pg-fimbriae. Similar results were obtained when whole cells of P. gingivalis were used as stimulus in lieu of purified fimbriae (not shown; see Fig. 5B for related experiment). Pg-Fimbriae Induce CXCR4-Mediated Activation of cAMP-Dependent PKA That Inhibits NF-␬B. Because the interaction of Pg-fimbriae with

CXCR4 inhibits NF-␬B activation and TNF-␣ production but up-regulates IL-10 (Fig. 2), we speculated that the inhibitory effects could be mediated by endogenously produced IL-10. However, upon Ab-mediated neutralization of IL-10, we did not observe significant reversal of the inhibitory effects (not shown). We then turned our attention to cAMP because inhibition of NF-␬B and TNF-␣ and concomitant augmentation of IL-10 are reminiscent of

the immunomodulatory action of cAMP-inducing enterotoxins, as we previously observed (17). We first examined whether Pg-fimbriae induce a CXCR4dependent cAMP response. Indeed, Pg-fimbriae significantly augmented intracellular cAMP levels in CHO-CD14/TLR2/CXCR4 cells but not in CHO-CD14/TLR2 cells (Fig. S2A). This was confirmed by using human monocytic THP-1 cells (Fig. S2B), or primary human monocytes and mouse macrophages (not shown). In THP-1 cells, which do express CXCR4 (Fig. S2C), Pg-fimbriae augmented the basal intracellular cAMP levels by almost 4-fold (Fig. S2B). This activity depended on CXCR4, because treatment with AMD3100 or anti-CXCR4 mAb diminished cAMP induction to baseline levels (Fig. S2B). Because PKA is a major downstream effector of cAMP, we next investigated whether Pg-fimbriae can activate PKA through interaction with CXCR4. For this purpose, human monocytes were stimulated with Pg-fimbriae with or without pretreatment with AMD3100, SQ22536 (cAMP synthesis inhibitor), H89 (PKA inhibitor), or chelerythrin (protein kinase C inhibitor; control). We found that Pg-fimbriae stimulate PKA activity ⬇3-fold over basal activity, although AMD3100 reversed this effect, suggesting its dependence on CXCR4 (Fig. S2D). SQ22536 showed a potent inhibitory effect confirming the cAMP dependence of PKA activation (Fig. S2D). H89 (but not chelerythrin) inhibited Pg-fimbria-induced PKA activity (Fig. S2D) confirming the specificity of the PKA assay. Having shown that Pg-fimbriae induce cAMP-dependent PKA activation via CXCR4, we next hypothesized that PKA inhibits Pg-fimbria-induced cell activation. If the hypothesis is true, we would expect to see enhanced Pg-fimbria-induced cell activation in the presence of PKA inhibitors. Indeed, the ability of Pg-fimbriae to activate NF-␬B in CHO-CD14/TLR2/CXCR4 cells was significantly up-regulated by inhibitors of cAMP synthesis (SQ22536) and of PKA activation (H89 and PKI 6–22) (Fig. 5A). These upregulatory effects were similar to CXCR4 blockade and the levels of NF-␬B activation were comparable with those seen in CHO-

Fig. 4. CXCR4 inhibits TLR2-induced NF-␬B 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-␬B reporter system. After 48 h, the cells were stimulated for 6 h with Pg-fimbriae (1 ␮g/ml). NF-␬B 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 ␮g/ml) or anti-CXCR4 (5 ␮g/ml). (C) NF-␬B 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-␬B 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). 13534 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0803852105


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Fig. 5. Inhibitors of cAMP and PKA reverse CXCR4-mediated suppression of NF-␬B activation. CHO cells were cotransfected with human CD14, TLR2, and CXCR4, and with NF-␬B reporter system. After 48 h, the transfectants were pretreated as indicated and stimulated for 6 h with Pg-fimbriae (1 ␮g/ml) (A) or whole cells of P. gingivalis (moi ⫽ 10:1) (B). The concentrations used were: 1 ␮g/ml AMD3100, 200 ␮M SQ22536, 10 ␮M H89, 1 ␮M chelerythrin, 1 ␮M PKI 6 –22 (peptide inhibitor of PKA), 1 ␮M KT5823 (peptide inhibitor of PKG; control). NF-␬B activation is reported as RLA and the horizontal lines indicate the level of NF-␬B 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-␬B 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-␬B activation by inducing inhibitory cAMPdependent PKA signaling.

P. gingivalis Exploits CXCR4 in Vitro and in Vivo to Promote Its Survival. The ability of P. gingivalis to inhibit cell activation through

interactions of its fimbriae with CXCR4 may promote its resistance to the host’s clearing mechanisms. This hypothesis was tested by using the mouse model in vitro and in vivo. Because the killing of P. gingivalis by mouse phagocytes is mediated by NO (18), we first determined whether CXCR4 inhibits induction of NO (measured as NO⫺ 2 , its stable metabolite) by P. gingivalis in mouse macro-

phages. Indeed, CXCR4 blockade with AMD3100 [highly specific antagonist of both human and mouse CXCR4 (19, 20)] or antiCXCR4 mAb resulted in significantly enhanced levels of NO⫺ 2 (Fig. 6A), the production of which depended heavily on TLR2 (Fig. 6A Inset). Therefore, TLR2 promotes, whereas CXCR4 inhibits, NO production in response to P. gingivalis, as seen with NF-␬B activation. Importantly, the observed up-regulation of NO production by CXCR4 blockade correlated with significant decrease (by ⬇3 log10 units) in viable P. gingivalis counts (CFU), as revealed by an intracellular survival assay (Fig. 6B). This suggests that engagement of CXCR4 by P. gingivalis promotes its intracellular survival, and we next hypothesized that cAMP-dependent PKA is the CXCR4 downstream effector responsible for this effect. The hypothesis was tested by using a similar intracellular survival assay performed in the presence of inhibitors of cAMP synthesis (SQ22536) and of PKA activation (H89 and PKI 6–22). We found that P. gingivalis-

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 ␮g/ml AMD3100, 15 ␮g/ml anti-CXCR4 mAb or isotype control, 200 ␮M SQ22536, 10 ␮M H89, 1 ␮M chelerythrin, 1 ␮M PKI 6 –22, and 1 ␮M 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 ␮g 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).



IMMUNOLOGY

CD14/TLR2 cells (Fig. 5A). In contrast, inhibitors of irrelevant kinases (chelerythrin or KT5823) had no effect (Fig. 5A). Similar results were obtained when the experiment was repeated using whole cells of P. gingivalis as the assay agonist (Fig. 5B). These data are consistent with a model according to which cAMP-dependent PKA acts as a CXCR4 downstream effector that inhibits TLR2dependent NF-␬B activation by P. gingivalis (Fig. 5C).

induced NO production and killing were up-regulated by treatments that inhibit cAMP synthesis and PKA activation, although not by inhibitors of irrelevant kinases (Fig. 6 C and D). Therefore, at least in vitro, P. gingivalis exploits CXCR4 for inhibiting NO production and promoting its survival and virulence. To determine whether this putative immune evasion mechanism operates in vivo, we next examined the ability of mice to clear i.p. infection with P. gingivalis, in the presence or absence of AMD3100, with or without N(G)-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of NO synthesis. We assessed viable P. gingivalis counts and production of NO in peritoneal lavage fluid collected at 20 h postinfection. We found that peritoneal fluid samples from AMD3100-treated mice contained ⬇2 log10 units more CFU compared with mice treated with PBS control (Fig. 6E), suggesting that CXCR4 blockade promotes P. gingivalis killing. However, this effect was dramatically reversed in AMD3100-treated mice that also received L-NAME (but not the inactive enantiomer DNAME) (Fig. 6E), suggesting that CXCR4 blockade promotes killing in a NO-dependent way. Assessment of NO levels in the peritoneal fluid confirmed that CXCR4 blockade up-regulates NO production, whereas L-NAME inhibits NO production (Fig. 6F). In conclusion, both in vitro and in vivo findings implicate CXCR4 as a receptor that is usurped by P. gingivalis to undermine the host’s ability to clear this pathogen. Discussion We have presented evidence for an immune evasion strategy of P. gingivalis that depends on the ability of its surface fimbriae to instigate functional TLR2/CXCR4 co-association in lipid rafts. In the ensuing cross-talk between the two receptors, cAMPdependent PKA acts as a CXCR4 downstream effector that inhibits TLR2-induced NF-␬B activation by P. gingivalis (see model in Fig. 5C). PKA-mediated inhibition of NF-␬B activation downstream of CXCR4 may explain why this receptor inhibits production of TNF-␣ as well as NO, the synthesis of which is also NF-␬Bdependent (21). In sharp contrast, engagement of CXCR4 by Pg-fimbriae up-regulates IL-10, consistent with the observation that its transcription is positively regulated by cAMP (22). The CXCR4dependent up-regulation of IL-10, which also inhibits NO synthesis (23), may contribute to the observed ability of P. gingivalis to resist NO-mediated clearance in vitro and in vivo. It should be noted that TLR2 is critical for the in vitro or in vivo innate response to P. gingivalis (3, 24), which expresses a diverse mixture of atypical LPS molecules, including species that trigger TLR2 signaling or weakly stimulate TLR4 but potently antagonize TLR4 activation by other stronger agonists (25). The importance, therefore, of TLR2 in P. gingivalis recognition may explain why manipulation of TLR2 signaling through CXCR4 by this pathogen causes a pronounced impairment of host defense function. However, treatment with AMD3100, a bicyclam drug that inhibits ligand binding to CXCR4 and downstream signaling without itself inducing signaling or causing receptor internalization (16), served as an effective counterstrategy to promote the killing of P. gingivalis. The concept that P. gingivalis can proactively manipulate the host response is also supported by a recent report that its LPS markedly up-regulates IL-1R-associated kinase-M, a negative regulator of TLR signaling (26). Thus, the pathogen appears to employ distinct mechanisms for escaping innate immune surveillance, which may contribute to the chronicity of periodontal infections. Previous work by some of the coauthors has identified CXCR4 as a component of TLR4-based receptor complexes involved in LPS recognition (14). Subsequent studies by the same group (27) and an independent team of investigators (28) have characterized the outcome of LPS-CXCR4 interactions. Triantafilou et al. (27) demonstrated direct binding of LPS to CXCR4, which positively up-regulated LPS-induced IL-6 production. On the other hand, Kishore et al. (28) found that the LPS-CXCR4 interaction suppresses TLR4-induced activation of NF-␬B, although the observed 13536 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0803852105

inhibition was not evident at relative high concentrations of LPS (ⱖ10 ng/ml). These authors suggested that CXCR4 raises the threshold for LPS-induced and TLR4-mediated activation of NF-␬B (28). The two studies do not necessarily contradict each other as to the role of CXCR4 in LPS-induced cell activation. First, Triantafilou et al. used LPS at 100 ng/ml, which is beyond the threshold identified by Kishore et al. Second, IL-6 is not regulated solely by NF-␬B because the IL-6 gene contains cAMP-responsive elements important for its transcriptional regulation (29), as is the case with IL-10 (22). In fact, we confirmed that forskolin (which elevates intracellular cAMP) as well as dibutyryl cAMP (membrane-permeable cAMP analog) synergize with LPS for induction of IL-6 production, whereas pharmacological inhibition of cAMPdependent PKA abrogates this effect (unpublished data). On the other hand, Pg-fimbriae interact with CXCR4 and inhibit NF-␬B activation over a wide concentration range [0.2–10 ␮g/ml, corresponding to 2 ⫻ 107 to 109 bacteria (30)]. CXCR4 can thus be considered as a negative regulator of Pg-fimbria-induced cell activation. Interestingly, the specificity of Pg-fimbriae for CXCR4 appears to be conferred by its FimCDE accessory components rather than by its major FimA subunit (unpublished data). Intriguingly, wild-type P. gingivalis is dramatically more virulent in an oral infection model than isogenic mutants expressing mutant fimbriae lacking the FimCDE components (10). This may suggest that the poor virulence of the mutants may, at least in part, be attributed to their inability to exploit CXCR4. Although the natural ligand of CXCR4 is the chemokine stromal cell-derived factor 1, HIV-1 also utilizes CXCR4 (15). Specifically, CXCR4 is an important coreceptor with CD4 for the HIV-1 envelope gp120/gp41 complex (15). AMD3100, which was found safe in human phase I clinical trials (31), has been successfully used to block CXCR4-dependent HIV-1 entry and replication (16, 32). Moreover, engagement of CXCR4 by HIV gp120 in T cells induces a hyporesponsive state attributable to cAMP-dependent PKA signaling, although this mechanism appears to be TLRindependent (33). We previously showed that macrophage uptake of P. gingivalis via complement receptor 3 leads to enhanced intracellular survival of the pathogen, attributable to the notion that this receptor is not linked to vigorous microbicidal mechanisms (10). We now know that P. gingivalis proactively manipulates the antimicrobial response of macrophages through CXCR4-mediated inhibition of NO production. These subversive activities, and the fact that P. gingivalis is resistant to NADPH oxidase-dependent killing (18), have the potential to prolong P. gingivalis infection and potentiate its impact on periodontitis and associated systemic diseases. The concept of microbial immune evasion constitutes a recurrent theme among successful pathogens, and CXCR4 appears to be exploited by at least two pathogens, HIV-1 (15) and P. gingivalis. The elucidation of specific mechanisms whereby pathogens undermine immunity is an essential prerequisite for developing counterstrategies to redirect the immune response to benefit host defense. Interestingly, CXCR4 expression is elevated in chronic periodontitis compared with healthy gingivae (34). Whether CXCR4 plays a role in periodontal disease has not been addressed, although such possibility is plausible because a predominant periodontal pathogen exploits CXCR4 for enhancing its virulence. Our demonstration that CXCR4 antagonism promotes P. gingivalis clearance holds promise for a potential therapeutic immunomodulation in human periodontitis and perhaps associated systemic diseases. Materials and Methods Reagents. SQ22536, H89, AMD3100, MCD, L-NAME, and D-NAME were purchased from Sigma–Aldrich. Chelerythrin, PKI 6 –22, and KT5823 were from Calbiochem. mAbs to human or mouse CXCR4 (12G5 and 247506, respectively) and isotype controls were from R&D Systems. mAbs to human or mouse TLR2 (TL2.1 and 6C2, respectively) and polyclonal anti-human/mouse CXCR4 were from eBioscience. mAbs to MHC class I (W6/32) and CD14 (Tu¨k 4) were from Abcam. For FRET


Cell Culture. Human monocytes were purified from peripheral blood (collected in compliance with established federal guidelines and institutional policies) upon centrifugation over NycoPrep 1.068, and incidental nonmonocytes were magnetically depleted (Miltenyi Biotec) (36). Purified monocytes were cultured at 37°C and 5% CO2 in RPMI medium 1640 (Invitrogen) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin G, 100 ␮g/ml streptomycin, and 0.05 mM 2-ME (complete RPMI). Complete RPMI was also used to culture THP-1 cells (ATCC TIB 202). CHO-K1 cells (ATCC CRL-9618) were maintained in Ham’s F-12 medium (Invitrogen) with 2 mM L-glutamine, 10% heatinactivated FBS, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Thioglycollate-elicited macrophages were isolated from the peritoneal cavity of wild-type or TLR2⫺/⫺ mice (The Jackson Laboratory) (3). Cell Transfections. By using PolyFect transfection reagent (Qiagen), CHO-K1 cells were transfected with human TLR2 and CD14, using a single plasmid (pDUOhCD14/TLR2; InvivoGen), with or without human CXCR4 (pORF-hCXCR4; InvivoGen). To monitor NF-␬B activation, the cells were cotransfected with NF-␬Bdependent firefly luciferase reporter plasmid (pNF-␬B-Luc; Stratagene) and a Renilla luciferase transfection control (pRLnull; Promega) (3). Cellular Activation Assays. Cytokine induction in stimulated cell culture supernatants was measured by ELISA (eBioscience). Levels of cAMP in activated cell extracts were measured by using a cAMP enzyme immunoassay kit (Cayman Chemical) (17). Induction of PKA activity was determined by using lysates of activated cells and the ProFluor PKA assay (Promega). Induction of NO production was assessed by measuring the amount of its stable metabolite NO2⫺ in stimulated culture supernatants or in peritoneal fluid by using a Griess reaction-based assay (R&D Systems). Cellular extracts were analyzed for NF-␬B p65 activation by using TransAM ELISA (Active Motif). NF-␬B-dependent transcription of a luciferase reporter gene was determined by measuring relative luciferase activity (RLA) in stimulated cells transfected with the NF-␬B reporter system described above. RLA was calculated as a ratio of firefly luciferase activity to Renilla luciferase activity, and results were normalized to those of unstimulated controls (3).

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FRET. Upon stimulation for 10 min at 37°C with Pg-fimbriae, human monocytes were labeled with a mixture of Cy3-conjugated mAb (donor) and Cy5-conjugated mAb (acceptor), as indicated in Fig. 1 A and C. The cells were washed and fixed, and energy transfer between various receptor pairs was calculated from the increase in donor fluorescence after acceptor photobleaching (3, 14). Binding Assays. The binding of FITC-labeled fimbriae to receptor-transfected cell lines was determined by using a fluorescent cell-based assay in 96-well plates, as described in refs. 13 and 35. Confocal Microscopy. To demonstrate colocalization of P. gingivalis with CXCR4 and TLR2, human monocytes or mouse macrophages were grown on chamber slides and exposed to FITC-labeled P. gingivalis for 10 min. The cells were then fixed, permeabilized, stained with Texas red-labeled anti-CXCR4 plus allophycocyanin-labeled anti-TLR2, and mounted with coverslips for imaging on an Olympus FV500 confocal microscope (10). Antibiotic Protection-Based Intracellular Survival Assay. The potential of P. gingivalis for intracellular survival was determined as described in ref. 10. Briefly, viable counts of internalized P. gingivalis were determined by plating serial dilutions of macrophage lysates on blood agar plates subjected to anaerobic culture. Before macrophage lysis, extracellular nonadherent bacteria were removed by washing, while extracellular adherent bacteria were killed by using gentamicin and metronidazole (10).

In Vivo Infection. BALB/c mice (8 –10 weeks old; The Jackson Laboratory) were pretreated with AMD3100 (i.p., 25 ␮g in 0.1 ml of PBS) or PBS alone. After 1 h, the mice were infected i.p. with P. gingivalis 33277 (5 ⫻ 107 CFU). Peritoneal lavage was performed 20 h postinfection. Serial 10-fold dilutions of peritoneal fluid were plated onto blood agar plates and cultured anaerobically for enumerating recovered peritoneal CFU. All animal procedures were performed in compliance with established federal guidelines and institutional policies. Statistical Analysis. Data were evaluated by ANOVA and the Dunnett multiplecomparison test (GraphPad InStat). Where appropriate, unpaired two-tailed t tests were performed. P ⬍ 0.05 was taken as the level of significance. ACKNOWLEDGMENTS. This work was supported by U.S. Public Health Service Grants DE015254, DE017138, and DE018292 (to G.H.) and by Sport Aiding Medical Research for Kids (to K.T.).

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IMMUNOLOGY

measurements, mAbs or cholera toxin B subunit (for labeling GM1; List Biological) were conjugated to Cy3 or Cy5 by using labeling kits from Amersham. P. gingivalis ATCC 33277 was grown anaerobically at 37°C in modified GAM medium (Nissui Pharmaceutical). LPS-free Pg-fimbriae were purified as described in ref. 35.