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 virtue of their length [up to 3 ␮m (8)], Pg-fimbriae may be the first (TLR)-mediated immunity that depends on CXCR4 exploitation. Spe- P. gingivalis molecule to interact with innate immune cells and cifically, the oral/systemic pathogen in- initiate intracellular signaling. The initial recognition event involves duces cross-talk between CXCR4 and TLR2 in human or binding of Pg-fimbriae to CD14, which serves as a coreceptor that mouse and undermines host defense. This is accom- facilitates TLR2 signaling (3, 11). The outcome of TLR2 activation plished through its surface fimbriae, which induce CXCR4/TLR2 co- in response to distinct microbial molecules may be influenced by association in rafts and interact with both receptors: Binding to differential TLR2 association with accessory receptors, as previ- CXCR4 induces cAMP-dependent kinase A (PKA) signaling, ously shown for TLR4 (12). Here, we have identified CXC- which in turn inhibits TLR2-mediated proinflammatory and antimi- chemokine receptor 4 (CXCR4) as a TLR2-associated receptor crobial responses to the pathogen. This outcome enables P. gingivalis interacting with Pg-fimbriae and examined a possible cross-talk to resist clearance in vitro and in vivo and thus to promote its adaptive between the two receptors. fitness. However, a specific CXCR4 antagonist abrogates this immune Strikingly, unlike CD14, which facilitates TLR2 activation by P. evasion mechanism and offers a promising counterstrategy for the gingivalis (3), CXCR4 appeared to limit TLR2 activation in human control of P. gingivalis periodontal or systemic infections. monocytes or mouse macrophages. Specifically, we found that Pg-fimbriae induce CXCR4-mediated activation of cAMP- ͉ ͉ ͉ ͉ bacterial pathogenesis immune evasion macrophages P. gingivalis dependent protein kinase A (PKA), which in turn inhibits TLR2- protein kinase A induced NF-␬B activation in response to P. gingivalis. CXCR4 may thus serve a homeostatic role to prevent excessive TLR2-induced icrobial infection is detected by pattern-recognition recep- or, alternatively, CXCR4 may be exploited by P. Mtors, among which Toll-like receptors (TLRs) play a central gingivalis for suppressing TLR2-mediated innate immunity. How- role in inducing innate immune responses for pathogen control (1). ever, we additionally found that the interaction of P. gingivalis with TLRs do not function in isolation but cooperate with other recep- CXCR4 impairs antimicrobial host defense and promotes the tors in multireceptor complexes within membrane lipid rafts (2–4). survival of the pathogen in vitro and in vivo. Therefore, P. gingivalis The formation of TLR-containing receptor clusters may serve to appears to exploit its interaction with CXCR4 as a mechanism of generate a combinatorial repertoire for discriminating among the immune evasion. abundant and diverse microbial molecules and thereby to tailor the host response. However, it is conceivable that may Results exploit the propensity of TLRs for cooperation with heterotypic Pg-Fimbriae Induce TLR2/CXCR4 Co-Association. Using FRET, we pre- receptors by instigating the recruitment of receptors that could viously showed that TLR2 is recruited to membrane lipid rafts and deregulate effective innate immunity. In this article, we present associates with CD14 in Pg-fimbria-activated monocytes but not evidence that Porphyromonas gingivalis effectively uses this immune when the rafts are disrupted by cholesterol depletion using methyl- evasion strategy. ␤-cyclodextrin (MCD) (3, 13). Using the same technique, we have P. gingivalis is a predominant pathogen associated with human now identified CXCR4 as a potential TLR2 coreceptor, in line with periodontitis, an infection-driven chronic inflammatory disease of earlier observations by some of the coauthors that this receptor is the oral cavity (5). This Gram-negative anaerobic organism is a component of pattern-recognition receptor complexes (14). Spe- moreover implicated in systemic conditions such as atherosclerosis cifically, significant energy transfer was detected between Cy3- (6) or aspiration pneumonia (7). The pathogenic potential of P. labeled TLR2 (donor) and Cy5-labeled CXCR4 (acceptor) in gingivalis is attributed to several virulence factors (e.g., fimbriae and stimulated but not in resting monocytes (Fig. 1A), indicating that cysteine proteinases), which enable the pathogen to colonize or invade host tissues and secure critical nutrients (8). However, a Pg-fimbriae induce TLR2/CXCR4 co-association. As expected, pathogen’s ability to find a niche and establish chronic infection Pg-fimbriae induced TLR2 association with CD14 (positive con- requires more than possessing appropriate adhesins or other factors trol) but not with MHC class I (negative control) (Fig. 1A). for nutrient procurement. To persist in a hostile host environment, pathogens should be able to evade or subvert the host immune Author contributions: G.H., M.T., and K.T. designed research; G.H., M.W., S.L., M.T., and K.T. system aiming to control or eliminate them. Successful pathogenic performed research; G.H., M.W., S.L., M.T., and K.T. analyzed data; and G.H. wrote the organisms that disable host defenses target preferentially innate paper. immunity (9). This may also undermine the overall host defense, The authors declare no conflict of interest. given the instructive role of innate immunity in the adaptive This article is a PNAS Direct Submission. B.A.B. is a guest editor invited by the Editorial immune response (1). Board. The fimbriae of P. gingivalis, which comprise polymerized fim- Freely available online through the PNAS open access option. brillin (FimA) and accessory (FimCDE) encoded by ‡To whom correspondence should be addressed at: University of Louisville, 501 South of the fimbrial operon (10), are traditionally recognized as a major Preston Street, Louisville, KY 40292. E-mail: [email protected]. colonization factor (8). In this article, we show that the fimbriae of This article contains supporting information online at www.pnas.org/cgi/content/full/ P. gingivalis (henceforth referred to as Pg-fimbriae) contribute to its 0803852105/DCSupplemental. virulence also through immune subversion of TLR signaling. By © 2008 by The National Academy of Sciences of the USA

13532–13537 ͉ PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803852105 Downloaded by guest on October 2, 2021 Fig. 1. CXCR4 associates with TLR2 in Pg-fimbria-activated cells. (A) Hu- man monocytes were pretreated or not with MCD (10 mM) and stimu- lated with Pg-fimbriae (1 ␮g/ml, 10 min). FRET between TLR2 (Cy3-la- beled) and CXCR4, CD14, or MHC class I (Cy5-labeled) was measured from the increase in donor (Cy3) fluores- cence after acceptor (Cy5) photo- bleaching. (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 (Up- per) or mouse macrophages (Lower). Data are means Ϯ SD (n ϭ 3). Asterisks show significant (P Ͻ 0.01) differ- ences vs. medium-only control. Black circles indicate significant (P Ͻ 0.01) reversal of FRET increase.

However, treatment of monocytes with MCD before stimulation To determine that Pg-fimbriae can indeed bind CXCR4, we abrogated the energy transfer between TLR2 and CXCR4 (Fig. used CHO-K1 cells that do not normally express CXCR4 (15) 1A), suggesting that their co-association takes place in lipid rafts. and, moreover, interact poorly with Pg-fimbriae (13). We thus To rule out that MCD causes loss or shedding of TLR2 or CXCR4, transfected CHO-K1 cells with human CXCR4 and examined we examined their expression in MCD-treated or untreated cells. the binding of biotinylated Pg-fimbriae probed with streptavidin- Indeed, flow cytometry revealed that MCD did not alter the FITC. Although Pg-fimbriae displayed poor binding to empty- expression of TLR2 or CXCR4 (Fig. 1B). Additional support for vector-transfected CHO-K1 cells, their binding was increased lipid raft association was obtained by demonstrating that CXCR4 Ͼ3-fold in CXCR4-transfected CHO-K1 cells (henceforth des- associates with an established lipid raft marker (GM1 ganglioside) ignated CHO-CXCR4 cells) (Fig. 3A). The interaction of fim- upon cell activation with Pg-fimbriae (Fig. 1C). Consistent with the briae with CHO-CXCR4 cells involved specific binding to notion that Pg-fimbriae induce TLR2/CXCR4 co-association, P. CXCR4 as shown by potent blocking effects of a specific gingivalis was found to colocalize with both CXCR4 and TLR2 in antagonist [AMD3100 (16)] and of anti-CXCR4 mAb, whereas human monocytes or mouse macrophages (Fig. 1D). We next isotype control or irrelevant mAb had no effect (Fig. 3A). The investigated the functional significance of TLR2/CXCR4 co- binding specificity was additionally confirmed by the finding that association in response to Pg-fimbriae. excess unlabeled Pg-fimbriae inhibited the binding of labeled Pg-fimbriae to CHO-CXCR4 cells (Fig. 3A). Incubation of Pg-Fimbriae Interact with CXCR4 and Suppress TLR2-Induced Cell Acti- increasing concentrations of ligand with CHO-CXCR4 or con- vation. The ability of Pg-fimbriae to activate monocytes depends trol CHO cells showed that Pg-fimbriae bind the former cells in almost exclusively on TLR2 (3). We tested the hypothesis that a saturable manner (Fig. 3B). In conclusion, the data from Figs. CXCR4 acts as a TLR2 coreceptor in Pg-fimbria-induced cell 2 and 3 are consistent with the intriguing notion that Pg-fimbriae activation. Specifically, we speculated that a blocking mAb to interact with CXCR4 and induce signaling that inhibits NF-␬B CXCR4 would suppress Pg-fimbria-induced cell activation. Sur- activation and TNF-␣ induction on the one hand but promotes prisingly, Pg-fimbriae induced significantly stronger NF-␬B activa- IL-10 production on the other. tion (Fig. 2A) and TNF-␣ production (Fig. 2B) in anti-CXCR4- To provide direct evidence that Pg-fimbriae induce CXCR4/ pretreated than in isotype-control-pretreated cells. Strikingly, the TLR2 cross-talk that suppresses TLR2-mediated NF-␬B activation, immunosuppressive IL-10 was conversely affected; i.e., it we performed the following experiment. CHO-K1 cells were trans- was down-regulated (Fig. 2C). These data indicate that CXCR4 fected with human CD14 and TLR2 to render them responsive to regulates Pg-fimbria-induced cell activation and imply that Pg- Pg-fimbriae in terms of NF-␬B activation [CHO-K1 cells express fimbriae interact directly with CXCR4. endogenous TLR1 and TLR6, either of which can be used by

Fig. 2. CXCR4 regulates human acti-

vation in response to Pg-fimbriae. Monocytes IMMUNOLOGY 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 Ϯ SD (n ϭ 3) from one of three independent sets of experiments yielding similar results. Asterisks show significant (P Ͻ 0.01) differences vs. IgG2a isotype and medi- um-only controls.

Hajishengallis et al. PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13533 Downloaded by guest on October 2, 2021 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 con- trol, irrelevant mAb (5 ␮g/ml), or 100-fold excess 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-associated fluorescence after staining with streptavidin (SA)-FITC. Data are means Ϯ SD (n ϭ 3) from typical experiments performed three (A) or two (B) times yielding similar findings. In A, the asterisk indicates significant increase in binding (P Ͻ 0.01 vs. vector control) and black circles denote significant (P Ͻ 0.01) inhibition of binding.

Pg-fimbriae for cooperative TLR2 signaling induction (3)]. A the immunomodulatory action of cAMP-inducing enterotoxins, as second group was additionally cotransfected with human CXCR4. we previously observed (17). Both CHO-CD14/TLR2 and CHO-CD14/TLR2/CXCR4 cells, as We first examined whether Pg-fimbriae induce a CXCR4- well as empty vector-transfected cells, were subsequently compared dependent cAMP response. Indeed, Pg-fimbriae significantly aug- for their potential to activate NF-␬B-dependent transcription in mented intracellular cAMP levels in CHO-CD14/TLR2/CXCR4 response to Pg-fimbriae. As expected, Pg-fimbriae could readily cells but not in CHO-CD14/TLR2 cells (Fig. S2A). This was activate NF-␬B in CHO-CD14/TLR2 cells (Fig. 4A). However, confirmed by using human monocytic THP-1 cells (Fig. S2B), or stimulation of CHO-CD14/TLR2/CXCR4 cells with Pg-fimbriae primary human monocytes and mouse macrophages (not shown). resulted in significant inhibition (59%) of NF-␬B activation (Fig. In THP-1 cells, which do express CXCR4 (Fig. S2C), Pg-fimbriae 4A), which was reversed upon CXCR4 blockade with AMD3100 or augmented the basal intracellular cAMP levels by almost 4-fold anti-CXCR4 mAb (Fig. 4B). Interestingly, similar experiments (Fig. S2B). This activity depended on CXCR4, because treatment performed in human embryonic kidney-293 cells revealed that with AMD3100 or anti-CXCR4 mAb diminished cAMP induction CXCR4 inhibits TLR2-induced cell activation by Pg-fimbriae re- to baseline levels (Fig. S2B). Because PKA is a major downstream gardless of whether TLR1 or TLR6 is cotransfected to serve as a effector of cAMP, we next investigated whether Pg-fimbriae can signaling partner [supporting information (SI) Fig. S1]. However, activate PKA through interaction with CXCR4. For this purpose, CXCR4 does not inhibit TLR2 signaling in a nonspecific way, human monocytes were stimulated with Pg-fimbriae with or with- because it did not affect TLR2/TLR1 or TLR2/TLR6 signaling by out pretreatment with AMD3100, SQ22536 (cAMP synthesis in- the lipopeptides Pam3Cys and MALP-2, respectively (Fig. S1). The hibitor), H89 (PKA inhibitor), or chelerythrin (protein kinase C up-regulatory effect of CXCR4 blockade on NF-␬B activation by inhibitor; control). We found that Pg-fimbriae stimulate PKA Pg fimbriae was observed over a wide concentration range of the activity Ϸ3-fold over basal activity, although AMD3100 reversed agonist (Fig. 4C). Therefore, these data strongly suggest that this effect, suggesting its dependence on CXCR4 (Fig. S2D). CXCR4 inhibits TLR2-dependent NF-␬B activation in response to SQ22536 showed a potent inhibitory effect confirming the cAMP Pg-fimbriae. Similar results were obtained when whole cells of P. dependence of PKA activation (Fig. S2D). H89 (but not chel- gingivalis were used as stimulus in lieu of purified fimbriae (not erythrin) inhibited Pg-fimbria-induced PKA activity (Fig. S2D) shown; see Fig. 5B for related experiment). confirming the specificity of the PKA assay. Having shown that Pg-fimbriae induce cAMP-dependent PKA Pg-Fimbriae Induce CXCR4-Mediated Activation of cAMP-Dependent activation via CXCR4, we next hypothesized that PKA inhibits PKA That Inhibits NF-␬B. Because the interaction of Pg-fimbriae with Pg-fimbria-induced cell activation. If the hypothesis is true, we CXCR4 inhibits NF-␬B activation and TNF-␣ production but would expect to see enhanced Pg-fimbria-induced cell activation in up-regulates IL-10 (Fig. 2), we speculated that the inhibitory effects the presence of PKA inhibitors. Indeed, the ability of Pg-fimbriae could be mediated by endogenously produced IL-10. However, to activate NF-␬B in CHO-CD14/TLR2/CXCR4 cells was signifi- upon Ab-mediated neutralization of IL-10, we did not observe cantly up-regulated by inhibitors of cAMP synthesis (SQ22536) and significant reversal of the inhibitory effects (not shown). We then of PKA activation (H89 and PKI 6–22) (Fig. 5A). These up- turned our attention to cAMP because inhibition of NF-␬B and regulatory effects were similar to CXCR4 blockade and the levels TNF-␣ and concomitant augmentation of IL-10 are reminiscent of 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 Ϯ SD (n ϭ 3) from one set of experiments that was repeated yielding similar findings. Asterisks show significant differences in NF-␬B activation (A and B, P Ͻ 0.01; C, P Ͻ 0.05). The controls against which comparisons were made were CHO-CD14/TLR2 cells (A), medium only (B), or IgG2a control (C).

13534 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803852105 Hajishengallis et al. Downloaded by guest on October 2, 2021 50 A B CHO-CD14/TLR2 C 45 ** CHO-CD14/TLR2 35 Pg * * * * Pg 40 30 * T CD14 Pretreatment L 35 Pretreatment * CXCR4 25 R 30 No inhibitor No inhibitor 2 25 AMD3100 20 AMD3100 20 SQ22536 15 SQ22536 B activation (RLA) H89 B activation (RLA) H89 15 proinflammatory / 10 Chelerythrin cAMP 10 Chelerythrin antimicrobial pathway NF- 5 PKI 6-22 NF- 5 PKI 6-22 KT5823 KT5823 PKA NF- B 0 0 expression

CHO cells transfected with CD14/TLR2 and CXCR4 CHO cells transfected with CD14/TLR2 and CXCR4

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 ϭ 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 Ϯ SD (nϭ3) of typical experiments performed three (A)or two (B) times yielding similar results. Asterisks show significant (P Ͻ 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 cAMP- dependent PKA signaling.

CD14/TLR2 cells (Fig. 5A). In contrast, inhibitors of irrelevant phages. Indeed, CXCR4 blockade with AMD3100 [highly specific kinases (chelerythrin or KT5823) had no effect (Fig. 5A). Similar antagonist of both human and mouse CXCR4 (19, 20)] or anti- Ϫ results were obtained when the experiment was repeated using CXCR4 mAb resulted in significantly enhanced levels of NO2 (Fig. whole cells of P. gingivalis as the assay agonist (Fig. 5B). These data 6A), the production of which depended heavily on TLR2 (Fig. 6A are consistent with a model according to which cAMP-dependent Inset). Therefore, TLR2 promotes, whereas CXCR4 inhibits, NO PKA acts as a CXCR4 downstream effector that inhibits TLR2- production in response to P. gingivalis, as seen with NF-␬B activa- dependent NF-␬B activation by P. gingivalis (Fig. 5C). tion. Importantly, the observed up-regulation of NO production by CXCR4 blockade correlated with significant decrease (by Ϸ3log10 P. gingivalis Exploits CXCR4 in Vitro and in Vivo to Promote Its units) in viable P. gingivalis counts (CFU), as revealed by an Survival. The ability of P. gingivalis to inhibit cell activation through intracellular survival assay (Fig. 6B). This suggests that engagement interactions of its fimbriae with CXCR4 may promote its resistance of CXCR4 by P. gingivalis promotes its intracellular survival, and we to the host’s clearing mechanisms. This hypothesis was tested by next hypothesized that cAMP-dependent PKA is the CXCR4 using the mouse model in vitro and in vivo. Because the killing of downstream effector responsible for this effect. The hypothesis was P. gingivalis by mouse phagocytes is mediated by NO (18), we first tested by using a similar intracellular survival assay performed in determined whether CXCR4 inhibits induction of NO (measured the presence of inhibitors of cAMP synthesis (SQ22536) and of Ϫ as NO2 , its stable metabolite) by P. gingivalis in mouse macro- 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. ϭ Ϫ gingivalis (moi 10:1). After 24 h, production of NO2 was assayed by the Griess reaction (A and C), and viable CFU of internalized were determined by using an Ϫ intracellular survival assay (B and D). (A Inset)NO2 pro- duction in P. gingivalis-stimulated wild-type or TLR2Ϫ/Ϫ 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. 7 gingivalis (5 ϫ 10 CFU). The administration of pretreat- IMMUNOLOGY ing agents was repeated 8 h postinfection. Peritoneal fluid was collected 20 h postinfection and used to deter- Ϫ 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 as means Ϯ SD (A–D, n ϭ 3; F, n ϭ 5) or are shown for each mouse with horizontal lines indicating mean values (E). The asterisks show significant (P Ͻ 0.01) differences vs. medium-only treatments (A–D), vs. wild-type macro- phages (A Inset), or vs. PBS-treated groups (E and F).

Hajishengallis et al. PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13535 Downloaded by guest on October 2, 2021 induced NO production and killing were up-regulated by treat- inhibition was not evident at relative high concentrations of LPS ments that inhibit cAMP synthesis and PKA activation, although (Ն10 ng/ml). These authors suggested that CXCR4 raises the not by inhibitors of irrelevant kinases (Fig. 6 C and D). Therefore, threshold for LPS-induced and TLR4-mediated activation of at least in vitro, P. gingivalis exploits CXCR4 for inhibiting NO NF-␬B (28). The two studies do not necessarily contradict each production and promoting its survival and virulence. other as to the role of CXCR4 in LPS-induced cell activation. First, To determine whether this putative immune evasion mechanism Triantafilou et al. used LPS at 100 ng/ml, which is beyond the operates in vivo, we next examined the ability of mice to clear i.p. threshold identified by Kishore et al. Second, IL-6 is not regulated infection with P. gingivalis, in the presence or absence of AMD3100, solely by NF-␬B because the IL-6 gene contains cAMP-responsive with or without N(G)-nitro-L-arginine methyl ester (L-NAME), a elements important for its transcriptional regulation (29), as is the specific inhibitor of NO synthesis. We assessed viable P. gingivalis case with IL-10 (22). In fact, we confirmed that forskolin (which counts and production of NO in peritoneal lavage fluid collected at elevates intracellular cAMP) as well as dibutyryl cAMP (mem- 20 h postinfection. We found that peritoneal fluid samples from brane-permeable cAMP analog) synergize with LPS for induction AMD3100-treated mice contained Ϸ2log10 units more CFU of IL-6 production, whereas pharmacological inhibition of cAMP- compared with mice treated with PBS control (Fig. 6E), suggesting dependent PKA abrogates this effect (unpublished data). On the that CXCR4 blockade promotes P. gingivalis killing. However, this other hand, Pg-fimbriae interact with CXCR4 and inhibit NF-␬B effect was dramatically reversed in AMD3100-treated mice that activation over a wide concentration range [0.2–10 ␮g/ml, corre- also received L-NAME (but not the inactive enantiomer D- sponding to 2 ϫ 107 to 109 bacteria (30)]. CXCR4 can thus be NAME) (Fig. 6E), suggesting that CXCR4 blockade promotes considered as a negative regulator of Pg-fimbria-induced cell killing in a NO-dependent way. Assessment of NO levels in the activation. Interestingly, the specificity of Pg-fimbriae for CXCR4 peritoneal fluid confirmed that CXCR4 blockade up-regulates NO appears to be conferred by its FimCDE accessory components production, whereas L-NAME inhibits NO production (Fig. 6F). In rather than by its major FimA subunit (unpublished data). Intrigu- conclusion, both in vitro and in vivo findings implicate CXCR4 as ingly, wild-type P. gingivalis is dramatically more virulent in an oral a receptor that is usurped by P. gingivalis to undermine the host’s infection model than isogenic mutants expressing mutant fimbriae ability to clear this pathogen. lacking the FimCDE components (10). This may suggest that the poor virulence of the mutants may, at least in part, be attributed to Discussion their inability to exploit CXCR4. We have presented evidence for an immune evasion strategy of P. Although the natural ligand of CXCR4 is the chemokine stromal gingivalis that depends on the ability of its surface fimbriae to cell-derived factor 1, HIV-1 also utilizes CXCR4 (15). Specifically, instigate functional TLR2/CXCR4 co-association in lipid rafts. In CXCR4 is an important coreceptor with CD4 for the HIV-1 the ensuing cross-talk between the two receptors, cAMP- envelope gp120/gp41 complex (15). AMD3100, which was found dependent PKA acts as a CXCR4 downstream effector that inhibits safe in human phase I clinical trials (31), has been successfully used ␬ TLR2-induced NF- B activation by P. gingivalis (see model in Fig. to block CXCR4-dependent HIV-1 entry and replication (16, 32). ␬ 5C). PKA-mediated inhibition of NF- B activation downstream of Moreover, engagement of CXCR4 by HIV gp120 in T cells induces CXCR4 may explain why this receptor inhibits production of a hyporesponsive state attributable to cAMP-dependent PKA ␣ ␬ TNF- as well as NO, the synthesis of which is also NF- B- signaling, although this mechanism appears to be TLR- dependent (21). In sharp contrast, engagement of CXCR4 by independent (33). Pg-fimbriae up-regulates IL-10, consistent with the observation that We previously showed that uptake of P. gingivalis via its transcription is positively regulated by cAMP (22). The CXCR4- complement receptor 3 leads to enhanced intracellular survival of dependent up-regulation of IL-10, which also inhibits NO synthesis the pathogen, attributable to the notion that this receptor is not (23), may contribute to the observed ability of P. gingivalis to resist linked to vigorous microbicidal mechanisms (10). We now know NO-mediated clearance in vitro and in vivo. that P. gingivalis proactively manipulates the antimicrobial response It should be noted that TLR2 is critical for the in vitro or in vivo of macrophages through CXCR4-mediated inhibition of NO pro- innate response to P. gingivalis (3, 24), which expresses a diverse duction. These subversive activities, and the fact that P. gingivalis is mixture of atypical LPS molecules, including species that trigger resistant to NADPH oxidase-dependent killing (18), have the TLR2 signaling or weakly stimulate TLR4 but potently antagonize potential to prolong P. gingivalis infection and potentiate its impact TLR4 activation by other stronger agonists (25). The importance, on periodontitis and associated systemic diseases. therefore, of TLR2 in P. gingivalis recognition may explain why The concept of microbial immune evasion constitutes a recurrent manipulation of TLR2 signaling through CXCR4 by this pathogen theme among successful pathogens, and CXCR4 appears to be causes a pronounced impairment of host defense function. How- exploited by at least two pathogens, HIV-1 (15) and P. gingivalis. ever, treatment with AMD3100, a bicyclam drug that inhibits ligand The elucidation of specific mechanisms whereby pathogens under- binding to CXCR4 and downstream signaling without itself induc- mine immunity is an essential prerequisite for developing counter- ing signaling or causing receptor internalization (16), served as an strategies to redirect the immune response to benefit host defense. effective counterstrategy to promote the killing of P. gingivalis. The Interestingly, CXCR4 expression is elevated in chronic periodon- concept that P. gingivalis can proactively manipulate the host titis compared with healthy gingivae (34). Whether CXCR4 plays response is also supported by a recent report that its LPS markedly a role in periodontal disease has not been addressed, although such up-regulates IL-1R-associated kinase-M, a negative regulator of possibility is plausible because a predominant periodontal pathogen TLR signaling (26). Thus, the pathogen appears to employ distinct exploits CXCR4 for enhancing its virulence. Our demonstration mechanisms for escaping innate immune surveillance, which may that CXCR4 antagonism promotes P. gingivalis clearance holds contribute to the chronicity of periodontal infections. promise for a potential therapeutic immunomodulation in human Previous work by some of the coauthors has identified CXCR4 periodontitis and perhaps associated systemic diseases. as a component of TLR4-based receptor complexes involved in LPS recognition (14). Subsequent studies by the same group (27) and an Materials and Methods independent team of investigators (28) have characterized the Reagents. SQ22536, H89, AMD3100, MCD, L-NAME, and D-NAME were purchased outcome of LPS-CXCR4 interactions. Triantafilou et al. (27) from Sigma–Aldrich. Chelerythrin, PKI 6–22, and KT5823 were from Calbiochem. demonstrated direct binding of LPS to CXCR4, which positively mAbs to human or mouse CXCR4 (12G5 and 247506, respectively) and isotype up-regulated LPS-induced IL-6 production. On the other hand, controls were from R&D Systems. mAbs to human or mouse TLR2 (TL2.1 and 6C2, Kishore et al. (28) found that the LPS-CXCR4 interaction sup- respectively) and polyclonal anti-human/mouse CXCR4 were from eBioscience. presses TLR4-induced activation of NF-␬B, although the observed mAbs to MHC class I (W6/32) and CD14 (Tu¨k 4) were from Abcam. For FRET

13536 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803852105 Hajishengallis et al. Downloaded by guest on October 2, 2021 measurements, mAbs or cholera toxin B subunit (for labeling GM1; List Biological) FRET. Upon stimulation for 10 min at 37°C with Pg-fimbriae, human monocytes were conjugated to Cy3 or Cy5 by using labeling kits from Amersham. P. gingivalis were labeled with a mixture of Cy3-conjugated mAb (donor) and Cy5-conjugated ATCC 33277 was grown anaerobically at 37°C in modified GAM medium (Nissui mAb (acceptor), as indicated in Fig. 1 A and C. The cells were washed and fixed, Pharmaceutical). LPS-free Pg-fimbriae were purified as described in ref. 35. and energy transfer between various receptor pairs was calculated from the increase in donor fluorescence after acceptor photobleaching (3, 14). Cell Culture. Human monocytes were purified from peripheral blood (collected in compliance with established federal guidelines and institutional policies) upon Binding Assays. The binding of FITC-labeled fimbriae to receptor-transfected cell centrifugation over NycoPrep 1.068, and incidental nonmonocytes were mag- lines was determined by using a fluorescent cell-based assay in 96-well plates, as described in refs. 13 and 35. netically depleted (Miltenyi Biotec) (36). Purified monocytes were cultured at 37°C and 5% CO2 in RPMI medium 1640 (Invitrogen) supplemented with 10% Confocal Microscopy. To demonstrate colocalization of P. gingivalis with CXCR4 heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin G, 100 ␮g/ml and TLR2, human monocytes or mouse macrophages were grown on chamber streptomycin, and 0.05 mM 2-ME (complete RPMI). Complete RPMI was also used slides and exposed to FITC-labeled P. gingivalis for 10 min. The cells were then to culture THP-1 cells (ATCC TIB 202). CHO-K1 cells (ATCC CRL-9618) were main- fixed, permeabilized, stained with Texas red-labeled anti-CXCR4 plus allophyco- tained in Ham’s F-12 medium (Invitrogen) with 2 mM L-glutamine, 10% heat- cyanin-labeled anti-TLR2, and mounted with coverslips for imaging on an Olym- ␮ inactivated FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. Thioglycol- pus FV500 confocal microscope (10). late-elicited macrophages were isolated from the peritoneal cavity of wild-type Ϫ Ϫ or TLR2 / mice (The Jackson Laboratory) (3). Antibiotic Protection-Based Intracellular Survival Assay. The potential of P. gingivalis for intracellular survival was determined as described in ref. 10. Briefly, Cell Transfections. By using PolyFect transfection reagent (Qiagen), CHO-K1 cells viable counts of internalized P. gingivalis were determined by plating serial were transfected with human TLR2 and CD14, using a single plasmid (pDUO- dilutions of macrophage lysates on blood agar plates subjected to anaerobic hCD14/TLR2; InvivoGen), with or without human CXCR4 (pORF-hCXCR4; Invivo- culture. Before macrophage lysis, extracellular nonadherent bacteria were re- Gen). To monitor NF-␬B activation, the cells were cotransfected with NF-␬B- moved by washing, while extracellular adherent bacteria were killed by using dependent firefly luciferase reporter plasmid (pNF-␬B-Luc; Stratagene) and a gentamicin and metronidazole (10). Renilla luciferase transfection control (pRLnull; Promega) (3). In Vivo Infection. BALB/c mice (8–10 weeks old; The Jackson Laboratory) were ␮ Cellular Activation Assays. Cytokine induction in stimulated cell culture super- pretreated with AMD3100 (i.p., 25 g in 0.1 ml of PBS) or PBS alone. After 1 h, the ϫ 7 natants was measured by ELISA (eBioscience). Levels of cAMP in activated cell mice were infected i.p. with P. gingivalis 33277 (5 10 CFU). Peritoneal lavage was performed 20 h postinfection. Serial 10-fold dilutions of peritoneal fluid extracts were measured by using a cAMP enzyme immunoassay (Cayman were plated onto blood agar plates and cultured anaerobically for enumerating Chemical) (17). Induction of PKA activity was determined by using lysates of recovered peritoneal CFU. All animal procedures were performed in compliance activated cells and the ProFluor PKA assay (Promega). Induction of NO production Ϫ with established federal guidelines and institutional policies. 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 Statistical Analysis. Data were evaluated by ANOVA and the Dunnett multiple- ␬ (R&D Systems). Cellular extracts were analyzed for NF- B p65 activation by using comparison test (GraphPad InStat). Where appropriate, unpaired two-tailed t ␬ TransAM ELISA (Active Motif). NF- B-dependent transcription of a luciferase tests were performed. P Ͻ 0.05 was taken as the level of significance. reporter gene was determined by measuring relative luciferase activity (RLA) in ␬ stimulated cells transfected with the NF- B reporter system described above. RLA ACKNOWLEDGMENTS. This work was supported by U.S. Public Health Service was calculated as a ratio of firefly luciferase activity to Renilla luciferase activity, Grants DE015254, DE017138, and DE018292 (to G.H.) and by Sport Aiding Medical and results were normalized to those of unstimulated controls (3). Research for Kids (to K.T.).

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