Pathogen induction of CXCR4/TLR2 cross-talk impairshost defense functionGeorge 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 LouisvilleHealth 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 reviewApril 23, 2008)

We report a mechanism of microbial evasion of Toll-like receptor(TLR)-mediated immunity that depends on CXCR4 exploitation. Spe-cifically, the oral/systemic pathogen Porphyromonas gingivalis in-duces cross-talk between CXCR4 and TLR2 in human monocytes ormouse macrophages and undermines host defense. This is accom-plished through its surface fimbriae, which induce CXCR4/TLR2 co-association in lipid rafts and interact with both receptors: Binding toCXCR4 induces cAMP-dependent protein kinase A (PKA) signaling,which in turn inhibits TLR2-mediated proinflammatory and antimi-crobial responses to the pathogen. This outcome enables P. gingivalisto resist clearance in vitro and in vivo and thus to promote its adaptivefitness. However, a specific CXCR4 antagonist abrogates this immuneevasion mechanism and offers a promising counterstrategy for thecontrol of P. gingivalis periodontal or systemic infections.

bacterial pathogenesis � immune evasion � macrophages � P. gingivalis �protein kinase A

Microbial infection is detected by pattern-recognition recep-tors, 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 recep-tors in multireceptor complexes within membrane lipid rafts (2–4).The formation of TLR-containing receptor clusters may serve togenerate a combinatorial repertoire for discriminating among theabundant and diverse microbial molecules and thereby to tailor thehost response. However, it is conceivable that pathogens mayexploit the propensity of TLRs for cooperation with heterotypicreceptors by instigating the recruitment of receptors that couldderegulate effective innate immunity. In this article, we presentevidence that Porphyromonas gingivalis effectively uses this immuneevasion strategy.

P. gingivalis is a predominant pathogen associated with humanperiodontitis, an infection-driven chronic inflammatory disease ofthe oral cavity (5). This Gram-negative anaerobic organism ismoreover 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 andcysteine proteinases), which enable the pathogen to colonize orinvade host tissues and secure critical nutrients (8). However, apathogen’s ability to find a niche and establish chronic infectionrequires more than possessing appropriate adhesins or other factorsfor nutrient procurement. To persist in a hostile host environment,pathogens should be able to evade or subvert the host immunesystem aiming to control or eliminate them. Successful pathogenicorganisms that disable host defenses target preferentially innateimmunity (9). This may also undermine the overall host defense,given the instructive role of innate immunity in the adaptiveimmune response (1).

The fimbriae of P. gingivalis, which comprise polymerized fim-brillin (FimA) and accessory proteins (FimCDE) encoded by genesof the fimbrial operon (10), are traditionally recognized as a majorcolonization factor (8). In this article, we show that the fimbriae ofP. gingivalis (henceforth referred to as Pg-fimbriae) contribute to itsvirulence also through immune subversion of TLR signaling. By

virtue of their length [up to 3 �m (8)], Pg-fimbriae may be the firstP. gingivalis molecule to interact with innate immune cells andinitiate intracellular signaling. The initial recognition event involvesbinding of Pg-fimbriae to CD14, which serves as a coreceptor thatfacilitates TLR2 signaling (3, 11). The outcome of TLR2 activationin response to distinct microbial molecules may be influenced bydifferential TLR2 association with accessory receptors, as previ-ously shown for TLR4 (12). Here, we have identified CXC-chemokine receptor 4 (CXCR4) as a TLR2-associated receptorinteracting with Pg-fimbriae and examined a possible cross-talkbetween the two receptors.

Strikingly, unlike CD14, which facilitates TLR2 activation by P.gingivalis (3), CXCR4 appeared to limit TLR2 activation in humanmonocytes or mouse macrophages. Specifically, we found thatPg-fimbriae induce CXCR4-mediated activation of cAMP-dependent protein kinase A (PKA), which in turn inhibits TLR2-induced NF-�B activation in response to P. gingivalis. CXCR4 maythus serve a homeostatic role to prevent excessive TLR2-inducedinflammation or, alternatively, CXCR4 may be exploited by P.gingivalis for suppressing TLR2-mediated innate immunity. How-ever, we additionally found that the interaction of P. gingivalis withCXCR4 impairs antimicrobial host defense and promotes thesurvival of the pathogen in vitro and in vivo. Therefore, P. gingivalisappears to exploit its interaction with CXCR4 as a mechanism ofimmune evasion.

ResultsPg-Fimbriae Induce TLR2/CXCR4 Co-Association. Using FRET, we pre-viously showed that TLR2 is recruited to membrane lipid rafts andassociates with CD14 in Pg-fimbria-activated monocytes but notwhen the rafts are disrupted by cholesterol depletion using methyl-�-cyclodextrin (MCD) (3, 13). Using the same technique, we havenow identified CXCR4 as a potential TLR2 coreceptor, in line withearlier observations by some of the coauthors that this receptor isa component of pattern-recognition receptor complexes (14). Spe-cifically, significant energy transfer was detected between Cy3-labeled TLR2 (donor) and Cy5-labeled CXCR4 (acceptor) instimulated but not in resting monocytes (Fig. 1A), indicating thatPg-fimbriae induce TLR2/CXCR4 co-association. As expected,Pg-fimbriae induced TLR2 association with CD14 (positive con-trol) but not with MHC class I (negative control) (Fig. 1A).

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 thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. B.A.B. is a guest editor invited by the EditorialBoard.

Freely available online through the PNAS open access option.

‡To whom correspondence should be addressed at: University of Louisville, 501 SouthPreston Street, Louisville, KY 40292. E-mail: g0haji01@louisville.edu.

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 stimulationabrogated the energy transfer between TLR2 and CXCR4 (Fig.1A), 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 theexpression of TLR2 or CXCR4 (Fig. 1B). Additional support forlipid raft association was obtained by demonstrating that CXCR4associates with an established lipid raft marker (GM1 ganglioside)upon cell activation with Pg-fimbriae (Fig. 1C). Consistent with thenotion that Pg-fimbriae induce TLR2/CXCR4 co-association, P.gingivalis was found to colocalize with both CXCR4 and TLR2 inhuman monocytes or mouse macrophages (Fig. 1D). We nextinvestigated the functional significance of TLR2/CXCR4 co-association in response to Pg-fimbriae.

Pg-Fimbriae Interact with CXCR4 and Suppress TLR2-Induced Cell Acti-vation. The ability of Pg-fimbriae to activate monocytes dependsalmost exclusively on TLR2 (3). We tested the hypothesis thatCXCR4 acts as a TLR2 coreceptor in Pg-fimbria-induced cellactivation. Specifically, we speculated that a blocking mAb toCXCR4 would suppress Pg-fimbria-induced cell activation. Sur-prisingly, Pg-fimbriae induced significantly stronger NF-�B activa-tion (Fig. 2A) and TNF-� production (Fig. 2B) in anti-CXCR4-pretreated than in isotype-control-pretreated cells. Strikingly, theimmunosuppressive cytokine IL-10 was conversely affected; i.e., itwas down-regulated (Fig. 2C). These data indicate that CXCR4regulates Pg-fimbria-induced cell activation and imply that Pg-fimbriae interact directly with CXCR4.

To determine that Pg-fimbriae can indeed bind CXCR4, weused CHO-K1 cells that do not normally express CXCR4 (15)and, moreover, interact poorly with Pg-fimbriae (13). We thustransfected CHO-K1 cells with human CXCR4 and examinedthe binding of biotinylated Pg-fimbriae probed with streptavidin-FITC. Although Pg-fimbriae displayed poor binding to empty-vector-transfected CHO-K1 cells, their binding was increased�3-fold in CXCR4-transfected CHO-K1 cells (henceforth des-ignated CHO-CXCR4 cells) (Fig. 3A). The interaction of fim-briae with CHO-CXCR4 cells involved specific binding toCXCR4 as shown by potent blocking effects of a specificantagonist [AMD3100 (16)] and of anti-CXCR4 mAb, whereasisotype control or irrelevant mAb had no effect (Fig. 3A). Thebinding specificity was additionally confirmed by the finding thatexcess unlabeled Pg-fimbriae inhibited the binding of labeledPg-fimbriae to CHO-CXCR4 cells (Fig. 3A). Incubation ofincreasing concentrations of ligand with CHO-CXCR4 or con-trol CHO cells showed that Pg-fimbriae bind the former cells ina saturable manner (Fig. 3B). In conclusion, the data from Figs.2 and 3 are consistent with the intriguing notion that Pg-fimbriaeinteract with CXCR4 and induce signaling that inhibits NF-�Bactivation and TNF-� induction on the one hand but promotesIL-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 trans-fected with human CD14 and TLR2 to render them responsive toPg-fimbriae in terms of NF-�B activation [CHO-K1 cells expressendogenous TLR1 and TLR6, either of which can be used by

Fig. 1. CXCR4 associates with TLR2in Pg-fimbria-activated cells. (A) Hu-man monocytes were pretreated ornot with MCD (10 mM) and stimu-lated with Pg-fimbriae (1 �g/ml, 10min). FRET between TLR2 (Cy3-la-beled) and CXCR4, CD14, or MHC classI (Cy5-labeled) was measured fromthe increase in donor (Cy3) fluores-cence after acceptor (Cy5) photo-bleaching. (B) MCD effect on TLR2 orCXCR4 surface expression using FACS.(C) Association of CXCR4 with GM1(lipid raft marker) in Pg-fimbria-activated monocytes, determined byFRET. (D) Confocal colocalization ofFITC-P. gingivalis with both CXCR4and TLR2 in human monocytes (Up-per) or mouse macrophages (Lower).Data are means � SD (n � 3). Asterisksshow significant (P � 0.01) differ-ences vs. medium-only control. Blackcircles indicate significant (P � 0.01)reversal of FRET increase.

Fig. 2. CXCR4 regulates human monocyte acti-vation in response to Pg-fimbriae. Monocyteswere stimulated with Pg-fimbriae with or withoutpretreatment with anti-CXCR4 mAb or isotypecontrol (5 �g/ml). After 90 min, cellular extractswere analyzed for NF-�B p65 activation (A). After16 h, culture supernatants were assayed for TNF-�(B) or IL-10 (C). Data are means � SD (n � 3) fromone of three independent sets of experimentsyielding similar results. Asterisks show significant(P � 0.01) differences vs. IgG2a isotype and medi-um-only controls.

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Pg-fimbriae for cooperative TLR2 signaling induction (3)]. Asecond group was additionally cotransfected with human CXCR4.Both CHO-CD14/TLR2 and CHO-CD14/TLR2/CXCR4 cells, aswell as empty vector-transfected cells, were subsequently comparedfor their potential to activate NF-�B-dependent transcription inresponse to Pg-fimbriae. As expected, Pg-fimbriae could readilyactivate NF-�B in CHO-CD14/TLR2 cells (Fig. 4A). However,stimulation of CHO-CD14/TLR2/CXCR4 cells with Pg-fimbriaeresulted in significant inhibition (59%) of NF-�B activation (Fig.4A), which was reversed upon CXCR4 blockade with AMD3100 oranti-CXCR4 mAb (Fig. 4B). Interestingly, similar experimentsperformed in human embryonic kidney-293 cells revealed thatCXCR4 inhibits TLR2-induced cell activation by Pg-fimbriae re-gardless of whether TLR1 or TLR6 is cotransfected to serve as asignaling 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 bythe lipopeptides Pam3Cys and MALP-2, respectively (Fig. S1). Theup-regulatory effect of CXCR4 blockade on NF-�B activation byPg fimbriae was observed over a wide concentration range of theagonist (Fig. 4C). Therefore, these data strongly suggest thatCXCR4 inhibits TLR2-dependent NF-�B activation in response toPg-fimbriae. Similar results were obtained when whole cells of P.gingivalis were used as stimulus in lieu of purified fimbriae (notshown; see Fig. 5B for related experiment).

Pg-Fimbriae Induce CXCR4-Mediated Activation of cAMP-DependentPKA That Inhibits NF-�B. Because the interaction of Pg-fimbriae withCXCR4 inhibits NF-�B activation and TNF-� production butup-regulates IL-10 (Fig. 2), we speculated that the inhibitory effectscould be mediated by endogenously produced IL-10. However,upon Ab-mediated neutralization of IL-10, we did not observesignificant reversal of the inhibitory effects (not shown). We thenturned our attention to cAMP because inhibition of NF-�B andTNF-� and concomitant augmentation of IL-10 are reminiscent of

the immunomodulatory action of cAMP-inducing enterotoxins, aswe previously observed (17).

We first examined whether Pg-fimbriae induce a CXCR4-dependent cAMP response. Indeed, Pg-fimbriae significantly aug-mented intracellular cAMP levels in CHO-CD14/TLR2/CXCR4cells but not in CHO-CD14/TLR2 cells (Fig. S2A). This wasconfirmed by using human monocytic THP-1 cells (Fig. S2B), orprimary human monocytes and mouse macrophages (not shown).In THP-1 cells, which do express CXCR4 (Fig. S2C), Pg-fimbriaeaugmented the basal intracellular cAMP levels by almost 4-fold(Fig. S2B). This activity depended on CXCR4, because treatmentwith AMD3100 or anti-CXCR4 mAb diminished cAMP inductionto baseline levels (Fig. S2B). Because PKA is a major downstreameffector of cAMP, we next investigated whether Pg-fimbriae canactivate PKA through interaction with CXCR4. For this purpose,human monocytes were stimulated with Pg-fimbriae with or with-out pretreatment with AMD3100, SQ22536 (cAMP synthesis in-hibitor), H89 (PKA inhibitor), or chelerythrin (protein kinase Cinhibitor; control). We found that Pg-fimbriae stimulate PKAactivity �3-fold over basal activity, although AMD3100 reversedthis effect, suggesting its dependence on CXCR4 (Fig. S2D).SQ22536 showed a potent inhibitory effect confirming the cAMPdependence of PKA activation (Fig. S2D). H89 (but not chel-erythrin) inhibited Pg-fimbria-induced PKA activity (Fig. S2D)confirming the specificity of the PKA assay.

Having shown that Pg-fimbriae induce cAMP-dependent PKAactivation via CXCR4, we next hypothesized that PKA inhibitsPg-fimbria-induced cell activation. If the hypothesis is true, wewould expect to see enhanced Pg-fimbria-induced cell activation inthe presence of PKA inhibitors. Indeed, the ability of Pg-fimbriaeto activate NF-�B in CHO-CD14/TLR2/CXCR4 cells was signifi-cantly up-regulated by inhibitors of cAMP synthesis (SQ22536) andof PKA activation (H89 and PKI 6–22) (Fig. 5A). These up-regulatory effects were similar to CXCR4 blockade and the levelsof NF-�B activation were comparable with those seen in CHO-

Fig. 3. Pg-fimbriae bind to CXCR4. (A)Empty vector- or CXCR4-transfected CHOcells were pretreated with AMD3100 (1 �g/ml), anti-CXCR4 mAb, IgG2a isotype con-trol, irrelevant mAb (5 �g/ml), or 100-foldexcess unlabeled fimbriae, and then incu-bated with biotinylated fimbriae (1 �g/ml).(B) Similar experiment, without inhibitors,using increasing concentrations of ligand.Binding was measured as cell-associatedfluorescence after staining with streptavidin (SA)-FITC. Data are means � SD (n � 3) from typical experiments performed three (A) or two (B) times yielding similarfindings. 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. CXCR4 inhibits TLR2-induced NF-�B activation in response to Pg-fimbriae. (A) CHO cells were transfected with human CD14 and TLR2 with or withoutCXCR4. 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 withPg-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 wasblocked 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 ofPg-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 similarfindings. 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 wereCHO-CD14/TLR2 cells (A), medium only (B), or IgG2a control (C).

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CD14/TLR2 cells (Fig. 5A). In contrast, inhibitors of irrelevantkinases (chelerythrin or KT5823) had no effect (Fig. 5A). Similarresults were obtained when the experiment was repeated usingwhole cells of P. gingivalis as the assay agonist (Fig. 5B). These dataare consistent with a model according to which cAMP-dependentPKA acts as a CXCR4 downstream effector that inhibits TLR2-dependent NF-�B activation by P. gingivalis (Fig. 5C).

P. gingivalis Exploits CXCR4 in Vitro and in Vivo to Promote ItsSurvival. The ability of P. gingivalis to inhibit cell activation throughinteractions of its fimbriae with CXCR4 may promote its resistanceto the host’s clearing mechanisms. This hypothesis was tested byusing the mouse model in vitro and in vivo. Because the killing ofP. gingivalis by mouse phagocytes is mediated by NO (18), we firstdetermined whether CXCR4 inhibits induction of NO (measuredas NO2

�, its stable metabolite) by P. gingivalis in mouse macro-

phages. Indeed, CXCR4 blockade with AMD3100 [highly specificantagonist of both human and mouse CXCR4 (19, 20)] or anti-CXCR4 mAb resulted in significantly enhanced levels of NO2

� (Fig.6A), the production of which depended heavily on TLR2 (Fig. 6AInset). Therefore, TLR2 promotes, whereas CXCR4 inhibits, NOproduction in response to P. gingivalis, as seen with NF-�B activa-tion. Importantly, the observed up-regulation of NO production byCXCR4 blockade correlated with significant decrease (by �3 log10

units) in viable P. gingivalis counts (CFU), as revealed by anintracellular survival assay (Fig. 6B). This suggests that engagementof CXCR4 by P. gingivalis promotes its intracellular survival, and wenext hypothesized that cAMP-dependent PKA is the CXCR4downstream effector responsible for this effect. The hypothesis wastested by using a similar intracellular survival assay performed inthe presence of inhibitors of cAMP synthesis (SQ22536) and ofPKA activation (H89 and PKI 6–22). We found that P. gingivalis-

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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 cellsof 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 (peptideinhibitor 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-�Bactivation in Pg-fimbria-stimulated CHO cells transfected with CD14 and TLR2 only. Data are means � SD (n�3) of typical experiments performed three (A) ortwo (B) times yielding similar results. Asterisks show significant (P � 0.01) up-regulation of NF-�B activation vs. no-inhibitor control. (C) Summarizing model ofthe data. Unlike CD14, which facilitates TLR2 activation by P. gingivalis, CXCR4 suppresses TLR2-mediated NF-�B activation by inducing inhibitory cAMP-dependent PKA signaling.

Fig. 6. CXCR4 blockade inhibits P. gingivalis survival invitro and in vivo in a NO-dependent way. (A–D) Mousemacrophages were treated with the indicated inhibitorsorcontrolsat theseconcentrations:1�g/mlAMD3100,15�g/ml anti-CXCR4 mAb or isotype control, 200 �MSQ22536, 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).After24h,productionofNO2

� wasassayed by the Griess reaction (A and C), and viable CFUof internalized bacteria were determined by using anintracellular survival assay (B and D). (A Inset) NO2

� pro-duction in P. gingivalis-stimulated wild-type or TLR2�/�macrophages. (EandF)BALB/cmice i.p.pretreatedornotwith AMD3100 (25 �g in 0.1 ml of PBS) with or withoutL-NAME or D-NAME (0.1 ml of 12.5 mM solution), asindicated. After 1 h, the mice were i.p. infected with P.gingivalis (5 � 107 CFU). The administration of pretreat-ing agents was repeated 8 h postinfection. Peritonealfluid was collected 20 h postinfection and used to deter-mine viable P. gingivalis CFU (E) and NO2

� production (F).Data are from one of two independent sets of experi-ments yielding similar findings, and are presented asmeans � SD (A–D, n � 3; F, n � 5) or are shown for eachmouse with horizontal lines indicating mean values (E).The asterisks show significant (P � 0.01) differences vs.medium-only treatments (A–D), vs. wild-type macro-phages (A Inset), or vs. PBS-treated groups (E and F).

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induced NO production and killing were up-regulated by treat-ments that inhibit cAMP synthesis and PKA activation, althoughnot by inhibitors of irrelevant kinases (Fig. 6 C and D). Therefore,at least in vitro, P. gingivalis exploits CXCR4 for inhibiting NOproduction and promoting its survival and virulence.

To determine whether this putative immune evasion mechanismoperates 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), aspecific inhibitor of NO synthesis. We assessed viable P. gingivaliscounts and production of NO in peritoneal lavage fluid collected at20 h postinfection. We found that peritoneal fluid samples fromAMD3100-treated mice contained �2 log10 units more CFUcompared with mice treated with PBS control (Fig. 6E), suggestingthat CXCR4 blockade promotes P. gingivalis killing. However, thiseffect was dramatically reversed in AMD3100-treated mice thatalso received L-NAME (but not the inactive enantiomer D-NAME) (Fig. 6E), suggesting that CXCR4 blockade promoteskilling in a NO-dependent way. Assessment of NO levels in theperitoneal fluid confirmed that CXCR4 blockade up-regulates NOproduction, whereas L-NAME inhibits NO production (Fig. 6F). Inconclusion, both in vitro and in vivo findings implicate CXCR4 asa receptor that is usurped by P. gingivalis to undermine the host’sability to clear this pathogen.

DiscussionWe have presented evidence for an immune evasion strategy of P.gingivalis that depends on the ability of its surface fimbriae toinstigate functional TLR2/CXCR4 co-association in lipid rafts. Inthe ensuing cross-talk between the two receptors, cAMP-dependent PKA acts as a CXCR4 downstream effector that inhibitsTLR2-induced NF-�B activation by P. gingivalis (see model in Fig.5C). PKA-mediated inhibition of NF-�B activation downstream ofCXCR4 may explain why this receptor inhibits production ofTNF-� as well as NO, the synthesis of which is also NF-�B-dependent (21). In sharp contrast, engagement of CXCR4 byPg-fimbriae up-regulates IL-10, consistent with the observation thatits transcription is positively regulated by cAMP (22). The CXCR4-dependent up-regulation of IL-10, which also inhibits NO synthesis(23), may contribute to the observed ability of P. gingivalis to resistNO-mediated clearance in vitro and in vivo.

It should be noted that TLR2 is critical for the in vitro or in vivoinnate response to P. gingivalis (3, 24), which expresses a diversemixture of atypical LPS molecules, including species that triggerTLR2 signaling or weakly stimulate TLR4 but potently antagonizeTLR4 activation by other stronger agonists (25). The importance,therefore, of TLR2 in P. gingivalis recognition may explain whymanipulation of TLR2 signaling through CXCR4 by this pathogencauses a pronounced impairment of host defense function. How-ever, treatment with AMD3100, a bicyclam drug that inhibits ligandbinding to CXCR4 and downstream signaling without itself induc-ing signaling or causing receptor internalization (16), served as aneffective counterstrategy to promote the killing of P. gingivalis. Theconcept that P. gingivalis can proactively manipulate the hostresponse is also supported by a recent report that its LPS markedlyup-regulates IL-1R-associated kinase-M, a negative regulator ofTLR signaling (26). Thus, the pathogen appears to employ distinctmechanisms for escaping innate immune surveillance, which maycontribute to the chronicity of periodontal infections.

Previous work by some of the coauthors has identified CXCR4as a component of TLR4-based receptor complexes involved in LPSrecognition (14). Subsequent studies by the same group (27) and anindependent team of investigators (28) have characterized theoutcome of LPS-CXCR4 interactions. Triantafilou et al. (27)demonstrated direct binding of LPS to CXCR4, which positivelyup-regulated LPS-induced IL-6 production. On the other hand,Kishore et al. (28) found that the LPS-CXCR4 interaction sup-presses TLR4-induced activation of NF-�B, although the observed

inhibition was not evident at relative high concentrations of LPS(�10 ng/ml). These authors suggested that CXCR4 raises thethreshold for LPS-induced and TLR4-mediated activation ofNF-�B (28). The two studies do not necessarily contradict eachother as to the role of CXCR4 in LPS-induced cell activation. First,Triantafilou et al. used LPS at 100 ng/ml, which is beyond thethreshold identified by Kishore et al. Second, IL-6 is not regulatedsolely by NF-�B because the IL-6 gene contains cAMP-responsiveelements important for its transcriptional regulation (29), as is thecase with IL-10 (22). In fact, we confirmed that forskolin (whichelevates intracellular cAMP) as well as dibutyryl cAMP (mem-brane-permeable cAMP analog) synergize with LPS for inductionof IL-6 production, whereas pharmacological inhibition of cAMP-dependent PKA abrogates this effect (unpublished data). On theother hand, Pg-fimbriae interact with CXCR4 and inhibit NF-�Bactivation over a wide concentration range [0.2–10 �g/ml, corre-sponding to 2 � 107 to 109 bacteria (30)]. CXCR4 can thus beconsidered as a negative regulator of Pg-fimbria-induced cellactivation. Interestingly, the specificity of Pg-fimbriae for CXCR4appears to be conferred by its FimCDE accessory componentsrather than by its major FimA subunit (unpublished data). Intrigu-ingly, wild-type P. gingivalis is dramatically more virulent in an oralinfection model than isogenic mutants expressing mutant fimbriaelacking the FimCDE components (10). This may suggest that thepoor virulence of the mutants may, at least in part, be attributed totheir inability to exploit CXCR4.

Although the natural ligand of CXCR4 is the chemokine stromalcell-derived factor 1, HIV-1 also utilizes CXCR4 (15). Specifically,CXCR4 is an important coreceptor with CD4 for the HIV-1envelope gp120/gp41 complex (15). AMD3100, which was foundsafe in human phase I clinical trials (31), has been successfully usedto block CXCR4-dependent HIV-1 entry and replication (16, 32).Moreover, engagement of CXCR4 by HIV gp120 in T cells inducesa hyporesponsive state attributable to cAMP-dependent PKAsignaling, although this mechanism appears to be TLR-independent (33).

We previously showed that macrophage uptake of P. gingivalis viacomplement receptor 3 leads to enhanced intracellular survival ofthe pathogen, attributable to the notion that this receptor is notlinked to vigorous microbicidal mechanisms (10). We now knowthat P. gingivalis proactively manipulates the antimicrobial responseof macrophages through CXCR4-mediated inhibition of NO pro-duction. These subversive activities, and the fact that P. gingivalis isresistant to NADPH oxidase-dependent killing (18), have thepotential to prolong P. gingivalis infection and potentiate its impacton periodontitis and associated systemic diseases.

The concept of microbial immune evasion constitutes a recurrenttheme among successful pathogens, and CXCR4 appears to beexploited by at least two pathogens, HIV-1 (15) and P. gingivalis.The elucidation of specific mechanisms whereby pathogens under-mine immunity is an essential prerequisite for developing counter-strategies to redirect the immune response to benefit host defense.Interestingly, CXCR4 expression is elevated in chronic periodon-titis compared with healthy gingivae (34). Whether CXCR4 playsa role in periodontal disease has not been addressed, although suchpossibility is plausible because a predominant periodontal pathogenexploits CXCR4 for enhancing its virulence. Our demonstrationthat CXCR4 antagonism promotes P. gingivalis clearance holdspromise for a potential therapeutic immunomodulation in humanperiodontitis and perhaps associated systemic diseases.

Materials and MethodsReagents. SQ22536,H89,AMD3100,MCD, L-NAME,and D-NAMEwerepurchasedfrom Sigma–Aldrich. Chelerythrin, PKI 6–22, and KT5823 were from Calbiochem.mAbs to human or mouse CXCR4 (12G5 and 247506, respectively) and isotypecontrols 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 (Tuk 4) were from Abcam. For FRET

13536 � www.pnas.org�cgi�doi�10.1073�pnas.0803852105 Hajishengallis et al.

measurements,mAbsorcholeratoxinBsubunit (for labelingGM1;ListBiological)wereconjugatedtoCy3orCy5byusing labelingkits fromAmersham.P.gingivalisATCC 33277 was grown anaerobically at 37°C in modified GAM medium (NissuiPharmaceutical). LPS-free Pg-fimbriae were purified as described in ref. 35.

Cell Culture. Human monocytes were purified from peripheral blood (collectedin compliance with established federal guidelines and institutional policies) uponcentrifugation over NycoPrep 1.068, and incidental nonmonocytes were mag-netically depleted (Miltenyi Biotec) (36). Purified monocytes were cultured at37°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/mlstreptomycin, and 0.05 mM 2-ME (complete RPMI). Complete RPMI was also usedto culture THP-1 cells (ATCC TIB 202). CHO-K1 cells (ATCC CRL-9618) were main-tained in Ham’s F-12 medium (Invitrogen) with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 �g/ml streptomycin. Thioglycol-late-elicited macrophages were isolated from the peritoneal cavity of wild-typeor TLR2�/� mice (The Jackson Laboratory) (3).

Cell Transfections. By using PolyFect transfection reagent (Qiagen), CHO-K1 cellswere transfected with human TLR2 and CD14, using a single plasmid (pDUO-hCD14/TLR2; InvivoGen), with or without human CXCR4 (pORF-hCXCR4; Invivo-Gen). To monitor NF-�B activation, the cells were cotransfected with NF-�B-dependent firefly luciferase reporter plasmid (pNF-�B-Luc; Stratagene) and aRenilla luciferase transfection control (pRLnull; Promega) (3).

Cellular Activation Assays. Cytokine induction in stimulated cell culture super-natants was measured by ELISA (eBioscience). Levels of cAMP in activated cellextracts were measured by using a cAMP enzyme immunoassay kit (CaymanChemical) (17). Induction of PKA activity was determined by using lysates ofactivatedcellsandtheProFluorPKAassay (Promega). InductionofNOproductionwas assessed by measuring the amount of its stable metabolite NO2

� in stimulatedculture 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 usingTransAM ELISA (Active Motif). NF-�B-dependent transcription of a luciferasereporter gene was determined by measuring relative luciferase activity (RLA) instimulated cells transfected with the NF-�B reporter system described above. RLAwas calculated as a ratio of firefly luciferase activity to Renilla luciferase activity,and results were normalized to those of unstimulated controls (3).

FRET. Upon stimulation for 10 min at 37°C with Pg-fimbriae, human monocyteswere labeledwithamixtureofCy3-conjugatedmAb(donor)andCy5-conjugatedmAb (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 theincrease in donor fluorescence after acceptor photobleaching (3, 14).

Binding Assays. The binding of FITC-labeled fimbriae to receptor-transfected celllines was determined by using a fluorescent cell-based assay in 96-well plates, asdescribed in refs. 13 and 35.

Confocal Microscopy. To demonstrate colocalization of P. gingivalis with CXCR4and TLR2, human monocytes or mouse macrophages were grown on chamberslides and exposed to FITC-labeled P. gingivalis for 10 min. The cells were thenfixed, permeabilized, stained with Texas red-labeled anti-CXCR4 plus allophyco-cyanin-labeled anti-TLR2, and mounted with coverslips for imaging on an Olym-pus 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 serialdilutions of macrophage lysates on blood agar plates subjected to anaerobicculture. Before macrophage lysis, extracellular nonadherent bacteria were re-moved by washing, while extracellular adherent bacteria were killed by usinggentamicin and metronidazole (10).

In Vivo Infection. BALB/c mice (8–10 weeks old; The Jackson Laboratory) werepretreated with AMD3100 (i.p., 25 �g in 0.1 ml of PBS) or PBS alone. After 1 h, themice were infected i.p. with P. gingivalis 33277 (5 � 107 CFU). Peritoneal lavagewas performed 20 h postinfection. Serial 10-fold dilutions of peritoneal fluidwere plated onto blood agar plates and cultured anaerobically for enumeratingrecovered peritoneal CFU. All animal procedures were performed in compliancewith established federal guidelines and institutional policies.

Statistical Analysis. Data were evaluated by ANOVA and the Dunnett multiple-comparison test (GraphPad InStat). Where appropriate, unpaired two-tailed ttests were performed. P � 0.05 was taken as the level of significance.

ACKNOWLEDGMENTS. This work was supported by U.S. Public Health ServiceGrantsDE015254,DE017138,andDE018292(toG.H.)andbySportAidingMedicalResearch for Kids (to K.T.).

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