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Pathogen Induction of CXCR4/TLR2 Cross-Talk Impairs Host Defense Function

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Pathogen Induction of CXCR4/TLR2 Cross-Talk Impairs Host Defense Function 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 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 Porphyromonas gingivalis in- initiate intracellular signaling. The initial recognition event involves duces cross-talk between CXCR4 and TLR2 in human monocytes or binding of Pg-fimbriae to CD14, which serves as a coreceptor that mouse macrophages 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 lipid rafts and interact with both receptors: Binding to differential TLR2 association with accessory receptors, as previ- CXCR4 induces cAMP-dependent protein 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- inflammation 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 pathogens 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 proteins (FimCDE) encoded by genes ‡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 September 25, 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
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