of March 17, 2018.This information is current as

Lipoprotein-Induced Tolerancebut Is Not Required for Bacterial ST2 Negatively Regulates TLR2 Signaling,

Jiang Huai WangJinghua Liu, Julliette M. Buckley, H. Paul Redmond and

http://www.jimmunol.org/content/184/10/5802doi: 10.4049/jimmunol.0904127April 2010;

2010; 184:5802-5808; Prepublished online 16J Immunol 

Referenceshttp://www.jimmunol.org/content/184/10/5802.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

ST2 Negatively Regulates TLR2 Signaling, but Is NotRequired for Bacterial Lipoprotein-Induced Tolerance

Jinghua Liu,1 Julliette M. Buckley,1 H. Paul Redmond, and Jiang Huai Wang

Activation of TLR signaling is critical for host innate immunity against bacterial infection. Previous studies reported that the ST2

receptor, a member of the Toll/IL-1 receptor superfamily, functions as a negative regulator of TLR4 signaling and maintains LPS

tolerance. However, it is undeterminedwhether ST2 negatively regulates TLR2 signaling and furthermore, whether a TLR2 agonist,

bacterial lipoprotein (BLP)-induced tolerance is dependent on ST2. In this study, we show that BLP stimulation-induced production

of proinflammatory cytokines and immunocomplex formation of TLR2–MyD88 and MyD88–IL-1R–associated kinase (IRAK)

were significantly enhanced in ST2-deficient macrophages compared with those in wild-type controls. Furthermore, overexpres-

sion of ST2 dose-dependently attenuated BLP-induced NF-kB activation, suggesting a negative regulatory role of ST2 in TLR2

signaling. A moderate but significantly attenuated production of TNF-a and IL-6 on a second BLP stimulation was observed in

BLP-pretreated, ST2-deficient macrophages, which is associated with substantially reduced IRAK-1 protein expression and

downregulated TLR2–MyD88 and MyD88–IRAK immunocomplex formation. ST2-deficient mice, when pretreated with a non-

lethal dose of BLP, benefitted from an improved survival against a subsequent lethal BLP challenge, indicating BLP tolerance

develops in the absence of the ST2 receptor. Taken together, our results demonstrate that ST2 acts as a negative regulator of TLR2

signaling, but is not required for BLP-induced tolerance. The Journal of Immunology, 2010, 184: 5802–5808.

The Toll/IL-1 receptor (TIR) superfamily, defined by thepresence of an intracellular TIRdomain, can be divided intotwomainsubgroups: theIL-1receptorsandtheTLRs.TLRs,

broadly distributed on immune cells, are the archetypal patternrecognition receptors and play a key role in mediating the signalingof downstream effectors in response to pathogen-associated mo-lecular patterns and in triggering the transcriptional inflammatoryresponse through activation of the transcription factor NF-kB,leading to the production of proinflammatory cytokines (1, 2). Thus,TLRs serve as innate sensors of pathogen attack and alert the bodyto the potential of bacterial infection. However, activation of TLRsis a double-edged sword (3). Although normally helping to eradicatepathogens from a local infection, a persistent activation of theTLR-mediated intracellular signal transduction pathway in mono-cytes/macrophages, characterized by the excessive release of pro-inflammatory cytokines, may lead to the development of septicshock syndrome.Pre-exposure to Gram-negative endotoxin/LPS induces a tran-

sient inhibition of TLR4 signaling to a secondary LPS challengewith diminished production of proinflammatory cytokines, therebyconferring protection against a subsequent “lethal” LPS challengeand resulting in a significant survival advantage (4, 5). This well-

established phenomenon is termed LPS tolerance. LPS tolerancemay represent a protective mechanism, whose primary function isto prevent excessive inflammatory response induced by over-activation of the TLR4 signaling pathway. However, acquisition ofLPS tolerance, characterized by downregulated proinflammatorycytokine production and upregulated anti-inflammatory proteinsynthesis, may contribute to an increased incidence of secondarybacterial infection in hospitalized patients due to development of animmune-suppressed state (4, 6).Tolerance toGram-positive bacterial cellwall components, such as

bacterial lipoprotein (BLP), a TLR2 agonist, is a lesser studied, al-though equally important, phenomenon (7). Our in vivo work dem-onstrated that pretreatment with a nonlethal dose of BLP protectsmice against not only a subsequent lethal BLP challenge, but alsoa subsequent lethal LPS challenge through a cross-tolerance to LPS(8). Unlike LPS tolerance, BLP tolerance-afforded protection againstmicrobial sepsis was observed in bothwild-type (WT) (8) and TLR4-deficient (9) mice. This is closely associated with BLP-induced re-programming in phagocytes characterized by hypo-responsiveness inproducing proinflammatory cytokines and simultaneously an en-hanced antimicrobial activity (7–9). Our in vitro work found thatBLP-induced tolerance is associated with suppressed TLR2 expres-sion and an inhibition in both MAP kinase phosphorylation and NF-kB activation (10). We further demonstrated that reduced IL-1R–associated kinase (IRAK)-1 expression and disassociated MyD88–IRAK immunocomplex formation contribute to the downregulatedTLR2 signaling pathway and the attenuated proinflammatory cyto-kine production observed in BLP-tolerized cells (11).The orphan receptor ST2, also known as T1 and DER4, is

a member of the TIR domain-containing superfamily (12). Incontrast to all of the well-characterized members of the TIR familythat induce the inflammatory response via activation of NF-kB,signaling through ST2 is unable to activate NF-kB although it canactivate MAPKs (13). ST2 is proven to be a selective marker of Th2cells (14), and promotes predominance of the Th2 response to in-flammatory stimuli, whereas suppressing the Th1 response (15, 16).More recently, studies have emerged on the role of ST2 in the

Department of Academic Surgery, University College Cork/National University ofIreland, Cork University Hospital, Cork, Ireland

1J.L. and J.M.B. contributed equally to this work.

Received for publication December 22, 2009. Accepted for publication March 9,2010.

This work was supported in part by the Science Foundation Ireland Research Fron-tiers Programme to J.H.W. (SFI/08/RFP/BIC1734).

Address correspondence and reprint requests to Dr. Jiang Huai Wang, Department ofAcademic Surgery, University College Cork/National University of Ireland, CorkUniversity Hospital, Cork, Ireland. E-mail address: jh.wang@ucc.ie

Abbreviations used in this paper: BLP, bacterial lipoprotein; BMDM, bone marrow-derived macrophages; EV, empty vector; IRAK, IL-1R–associated kinase; KO,knock-out; LTA, lipoteichoic acid; pAb, polyclonal Ab; PGN, peptidoglycan; TIR,Toll/IL-1 receptor; WT, wild-type.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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macrophage-mediated proinflammatory response. It has been re-ported that ST2 negatively regulates TLR4 signaling and attenuatesLPS-stimulated proinflammatory cytokine production (17, 18),either by inhibiting IkBa degradation (18) or by sequestrating theadaptor proteins MyD88 and Mal through its TIR domain (19). Asa result, ST2-deficient macrophages produce increased proin-flammatory cytokines in response to LPS stimulation (19). Moreimportantly, ST2 has been demonstrated to function as a crucialmediator for inducing LPS tolerance as both ST2-deficient mac-rophages and mice failed to develop LPS tolerance (19).In the current study,we report that ST2 negatively regulatesTLR2

signaling, as ST2-deficient macrophages produced substantiallyenhancedproinflammatory cytokines in response to stimulationwiththe TLR2 agonists, and overexpression of ST2 strongly attenuatedBLP-induced NF-kB activation. Furthermore, BLP-pretreated,ST2-deficient macrophages displayed a moderate but significantlyreduced production of TNF-a and IL-6 on a second BLP stimula-tion, whereas pretreatment with a nonlethal dose of BLP conferreda survival advantage in ST2-deficient mice against a subsequentlethal septic challenge. These results demonstrate that BLP-induced tolerance develops both in vitro and in vivo in the absenceof the ST2 receptor.

Materials and MethodsReagents and Abs

The TLR2 agonist BLP, a synthetic bacterial lipopeptide (Pam3Cys-Ser-Lys4-OH), was purchased from EMC Microcollections (Tubingen, Ger-many) and was endotoxin free as confirmed by the Limulus amebocytelysate assay (Charles River Endosafe, Charleston, SC). The other twoTLR2 agonists, lipoteichoic acids (LTA) and peptidoglycan (PGN) werefrom Sigma-Aldrich (St. Louis, MO). Abs that recognize TLR2, MyD88,and IRAK-1 were purchased from Santa Cruz Biotechnology (Santa Cruz,CA) and Abcam (Cambridge, MA), respectively. The expression vectorspCMV-Flag-ST2, pCMV-Flag-MyD88, and pcDNA-Flag-IRAK-1 werekindly provided by Professor Luke O’Neill (Trinity College Dublin,Dublin, Ireland). All plasmid DNA were isolated and purified with anEndo-free Plasmid Maxi Kit (Qiagen, Chatsworth, CA). All culture me-dium and reagents used for cell cultures were purchased from InvitrogenLife Technologies (Paisley, Scotland, U.K.). All other chemicals, unlessindicated, were from Sigma-Aldrich.

Mice and lethal BLP challenge in vivo

The 6- to 8-wk-old ST2-deficient mice that were backcrossed to the BALB/cbackground for eight generations and BALB/c mice were maintained in theUniversity Biological Services Unit, University College Cork. Mice werehoused in barrier cages under controlled environmental conditions (12/12 hof light/dark cycle, 55% 6 5% humidity, 23˚C) and had free access tostandard laboratory chow and water. Animals were fasted 12 h beforeexperiments and allowed water ad libitum. All animal procedures wereconducted in the University Biological Services Unit under a license fromthe Department of Health (Republic of Ireland). BALB/c mice were pur-chased from Harlan (Oxon, U.K.), and ST2-deficient mice were originallyprovided by Dr. Andrew McKenzie (Medical Research Council Laboratoryof Molecular Biology, Cambridge, U.K.).

Age- and weight-matched WT and ST2-deficient mice were injected i.p.with 10 mg/kg BLP to induce BLP tolerance (BLP-tolerized), as describedpreviously (8, 9). Nontolerized mice received an equal volume (200 ml) ofi.p. PBS (naive). Twenty-four hours later, both naive and BLP-tolerizedmice were challenged with a lethal dose of BLP at 45 mg/kg. Survival wasmonitored for at least 7 d.

Cells and cultures

HEK-hTLR2 cells stably transfected with a human TLR2 cDNA constructwere a gift from Dr. Evelyn A. Kurt-Jones (University of MassachusettsMedical School, Worcester, MA) and were maintained in DMEM supple-mented with 10% heat-inactivated FCS and G418 (0.5 mg/ml) at 37˚C ina humidified 5% CO2 atmosphere. Peritoneal macrophages were collectedfrom WT and ST2-deficient mice by peritoneal lavage and incubated withDMEM containing 10% heat-inactivated FCS in 24-well plates (Falcon,Lincoln Park, NJ) for 90 min to remove nonadherent cells, as described pre-viously (9). Bone marrow-derived macrophages (BMDM) were isolated from

the femurs of WT and ST2-deficient mice and cultured in DMEM containing20% heat-inactivated FCS, penicillin (100 U/ml), streptomycin sulfate (100mg/ml), and supplemented with 10 ng/ml recombinant mouse macrophage-CSF (R&D Systems, Minneapolis, MN) for 7 d at 37˚C in a humidified 5%CO2 atmosphere, as described previously (20). The purity of both peritonealmacrophages and BMDM was.95%, as confirmed by FACScan analysis ofthe positive F4/80 Ag staining with an anti-F4/80 Ab (Serotec, Oxford, U.K.).

Cytokine measurement

Peritoneal macrophages from WT and ST2-deficient mice were plated into24-well plates (23 105 cells/well) and stimulated with various doses of theTLR2 agonists BLP, LTA, and PGN for 16 h. For in vitro BLP toleranceexperiments, WT and ST2-deficient peritoneal macrophages in 24-wellplates (2 3 105 cells/well) were pretreated with various doses of BLP (0–100 ng/ml) for 24 h, and further stimulated with 1000 ng/ml BLP for 16 h.Cell-free supernatants were collected and stored at 280˚C until analysis.For in vivo BLP tolerance experiments, naive and BLP-tolerized WT andST2-deficient mice received a lethal dose of BLP (45 mg/kg), and bloodsamples were collected at 90 min and 4 h postseptic challenge. TNF-a andIL-6 concentrations in the supernatants or in the serum were assessed bycytometric bead array (BD Biosciences, San Jose, CA).

Transient transfection

HEK-hTLR2 cells were plated into 96-well plates (Falcon) at 33 104 cells/well, and transfected with 50 ng NF-kB-driven firefly luciferase plasmid(pNF-kB-Luc) (Clontech,MountainView, CA) and 1 ngHSV-TKpromoter-driven Renilla luciferase plasmid (pGL4.74) (Promega, Madison, WI), withLipofectamine 2000 (Invitrogen). These cells were either cotransfected withthe plasmid encoding ST2 at concentrations ranging from 25–75 ng andfurther stimulated with 100 ng/ml BLP for 6 h, or cotransfected with theplasmid encoding ST2 (0–50 ng), together with the plasmids encoding eitherMyD88 (25 ng) or IRAK-1 (25 ng). In all cases the amount of DNA trans-fected was kept constant by the addition of various amounts of the appro-priate empty vector plasmid. Luciferase activity was determined using thedual-luciferase reporter assay system (Promega). Transfection efficiencywas normalized in all experiments with simultaneous measurement of Re-nilla luciferase activities.

Immunoblotting and immunoprecipitation

After BLP stimulation, naive and BLP-tolerized BMDM isolated from WTand ST2-deficient mice were collected at various time periods, washed withice-cold PBS, and lysed on ice in cell lysis buffer (Cell SignalingTechnology,Beverly, MA), supplemented with 1 mM phenylmethylsulfonyl fluoride andprotease inhibitor mixture (Roche, Indianapolis, IN). The resultant lysateswere centrifuged and supernatants containing the cytoplasmic proteins werecollected. Protein concentrationwas determined using amicro bicinchoninicacid protein assay (Pierce, Rockford, IL). Equal amounts of protein extractswere separated on SDS-polyacrylamide gels and trans-blotted onto poly-vinylidene difluoride membranes (Schleicher & Schuell, Dassel, Germany).The membrane was blocked for 1 h at room temperature with PBS con-taining 0.05% Tween-20 and 5% nonfat milk, and probed overnight at 4˚Cwith primary Abs. Blots were then incubated with appropriate HRP-conjugated secondary Abs (Dako, Cambridge, U.K.) at room temperaturefor 1 h, developed with SuperSignal chemiluminescent substrate (Pierce),and captured with LAS-3000 imaging system (Fujifilm, Tokyo, Japan). Forimmunoprecipitation, equal amounts of extracted protein were incubatedwith anti-MyD88 polyclonal Ab (pAb) overnight at 4˚C on a rotator. There-after, 50 ml of a 50% slurry of prewashed protein A/G-agarose beads (Pierce)was added to each sample and incubated at 4˚C for an additional 2 h period.The samples were spun briefly and washed three times in the lysis buffer.Loading buffer was added to each sample and boiled for 10 min. The sampleswere then separated by SDS-polyacrylamide gels, trans-blotted onto poly-vinylidene difluoride membranes, and probed with anti-TLR2 pAb and anti–IRAK-1 pAb, respectively.

Statistical analysis

All data are expressed as the mean 6 SD. Significance analysis was per-formed using the log-rank test for survival and ANOVA for all others, withGraphPad software (Prism, La Jolla, CA). Differences were judged sta-tistically significant when the p value was ,0.05.

ResultsST2 negatively regulates TLR2 signaling

We first examined whether ST2-deficient macrophages produceincreased proinflammatory cytokines in response to stimulation

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with the TLR2 agonists. Peritoneal macrophages from WT andST2-deficient mice were stimulated with various doses of BLP,LTA, and PGN for 16 h to assess TNF-a and IL-6 production.Significantly increased TNF-a (Fig. 1A) and IL-6 (Fig. 1B) releasewas observed in BLP-stimulated, ST2-deficient macrophageswhen compared with BLP-stimulated, WT macrophages (p ,0.05), which occurs in a BLP dose-dependent manner. Similarly,stimulation with the other two TLR2 agonists, LTA and PGN,resulted in an enhanced production of TNF-a and IL-6 in ST2-deficient macrophages (p , 0.05 versus WT macrophages) (Fig.1). BMDM from ST2-deficient mice also produced more proin-flammatory cytokines than did cells from WT mice in response tostimulation with the TLR2 agonists (data not shown).Engagement ofTLR2byBLP triggersTLR2,MyD88, and IRAK-

1 activation, and promotes these upstream signaling components toform immunocomplexes, a process that plays a crucial role in BLPsignal transduction (10, 11, 21, 22). To further investigate the in-fluence of ST2 on BLP-stimulated TLR2 signaling, the expressionof, and the immunocomplex formation of TLR2, MyD88, andIRAK-1 in response to BLP stimulation were assessed in BMDMisolated from WT and ST2-deficient mice. Stimulation of WTBMDM with BLP resulted in a reduced expression of IRAK-1protein (Fig. 2A, 2C) and an enhanced formation of both TLR2–MyD88 and MyD88–IRAK immunocomplexes (Fig. 2B, 2D, 2E),indicating that BLP stimulation induces degradation of IRAK-1 andpromotes associations between TLR2, MyD88, and IRAK-1. AfterBLP stimulation ST2-deficient BMDM also displayed rapid deg-radation of IRAK-1 protein at similar levels to those of WTBMDM, although a slightly more reduced IRAK-1 expression at 10min post-BLP stimulation was observed in ST2-deficient BMDM(Fig. 2A, 2C). However, there was increased recruitment of MyD88to TLR2 and IRAK-1 to MyD88 to form TLR2–MyD88 andMyD88–IRAK immunocomplexes in response to BLP stimulationin ST2-deficient BMDM, as assessed by immunoprecipitation (Fig.2B, 2D, 2E). These results suggest that ST2 exerts an inhibitoryaction on BLP-stimulated TLR2 signaling.If ST2 has a negative regulatory effect on TLR2 signaling,

overexpression of STmay attenuateTLR2-mediated activation of itsdownstream signaling pathway. To test this, we transfected HEK-hTLR2 cells with various amounts of plasmid encoding ST2 andfurther stimulated the cells with 100 ng/ml BLP for 6 h. Stimulationof HEK-hTLR2 cells with BLP resulted in an ∼20-fold increase inNF-kB activation when compared with unstimulated cells; how-ever, overexpression of ST2 dose-dependently suppressed NF-kBactivation induced by BLP stimulation (Fig. 3A). Next, we soughtto examine whether ST2 affects overexpression of either MyD88 orIRAK-1 induced activation of their downstream signaling pathway.

Overexpression of MyD88 in HEK-hTLR2 cells led to a nearly120-fold increase in NF-kB activation; however, cotransfection ofST2 inhibited MyD88-induced NF-kB activation in a dose-dependent manner (Fig. 3B). In contrast, cotransfection of ST2failed to attenuate NF-kB activation induced by overexpression ofIRAK-1 (Fig. 3C), indicating ST2 negatively regulating TLR2signaling pathway upstream rather than downstream of IRAK-1.

BLP tolerance is induced in both ST2-deficient macrophagesand mice

ST2 has been demonstrated to function as a crucial mediator forinducing LPS tolerance, as both ST2-deficient macrophages andmice fail to developLPS tolerance (19); however, it is undeterminedwhether ST2 is also responsible for BLP-induced tolerance. Wefirst clarified the function of ST2 in BLP-induced tolerance in vitro.Pretreatment with BLP at 10 and 100 ng/ml for 24 h dose-dependently induced BLP tolerance in WT peritoneal macro-phages, with significantly reduced production of proinflammatorycytokines TNF-a and IL-6 in response to a second BLP stimulationat 1000 ng/ml (Fig. 4). Notably, a moderate but significantly at-tenuated release of TNF-a and IL-6 after a second BLP stimulationwas observed in BLP-pretreated, ST2-deficient macrophages whencompared with naive, ST2-deficient macrophages (p , 0.05) (Fig.4), indicating that BLP tolerance is induced in ST2-deficientmacrophages. BLP-pretreated, ST2-deficient BMDM also ex-hibited a substantial reduction in proinflammatory cytokine pro-duction in response to a second BLP stimulation (data not shown).Nevertheless, levels of the reduced proinflammatory cytokine re-lease seen in BLP-pretreated, ST2-deficient macrophages were stillhigher than those observed in BLP-tolerized, WT macrophages,suggesting that ST2 may play a minor contributory role in BLP-induced tolerance.To further validate the above finding in vivo, WT, and ST2-

deficientmice received either PBS (naive) orBLPat a nonlethal doseof 10 mg/kg (BLP-tolerized) 24 h before a lethal BLP challenge (45mg/kg). Consistent with our previous work (8), induction of BLPtolerance in WT mice protected these mice against a second lethalBLP challenge, with a significant decrease inmortality from 72% innaive, WT mice to 11% in BLP-tolerized, WT mice (p = 0.0002)(Fig. 5A). More importantly, a significant survival advantage aftera second lethal BLP challenge was observed in ST2-deficient micepretreated with a nonlethal dose of BLP, with an improved survivalfrom 19% in naive, ST2-deficient mice to 67% (p = 0.0008) (Fig.5A). Although BLP-tolerized, ST2-deficient mice exhibited a re-duced survival advantage compared with BLP-tolerized, WT mice(67 versus 89% of survival rates), this did not reach a statisticalsignificance (p = 0.1015). Similar to the significantly attenuated

FIGURE 1. Increased proinflammatory cytokine

release from ST2-deficient macrophages in response

to stimulation with the TLR2 agonists. Peritoneal

macrophages isolated from WT and ST2-deficient

mice were incubated with various doses of BLP,

LTA, or PGN for 16 h. TNF-a (A) and IL-6 (B)

concentrations in the culture supernatants were as-

sessed by cytometric bead array. Data are expressed

as the mean 6 SD of duplicate samples and repre-

sentative of at least four to six separate experiments.

pp , 0.05 compared with WT macrophages.

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proinflammatory cytokine production in BLP-tolerized, WT mice,pretreatment with a low dose of BLP in ST2-deficient mice also ledto a substantial reduction in serum TNF-a and IL-6 levels aftera second BLP challenge when compared with naive, ST2-deficientmice (p, 0.05) (Fig. 5B). These results indicate that BLP toleranceis also induced in ST2-deficient mice. We noted that naive, ST2-deficient mice had a slight higher mortality rate (81%) than didnaive, WT mice (72%), but there was no significant differencein survival between ST2-deficient and WT mice (p = 0.4166)(Fig. 5A). Furthermore, although ST2-deficient mice displayeda much higher level of serum IL-6, levels of serum TNF-a wereidentical between ST2-deficient and WT mice (Fig. 5B).Our previous work has shown that induction of BLP tolerance is

associated with a reprogramming of TLR2 signaling (7, 10). Inparticular, reduced IRAK-1 protein expression and disassociatedMyD88–IRAK immunocomplex formation are two critical molec-ular events in this reprogrammed signal transduction pathway (11).To further elucidate the molecular mechanisms underlying the de-velopment of BLP-induced tolerance in ST2-deficient macrophagesand mice, cytoplasmic proteins extracted from WT and ST2-de-ficient BMDM, pretreated with 100 ng/ml of BLP for 24 h andfurther stimulated with 1000 ng/ml BLP for different periods, weresubjected to immunoblotting and immunoprecipitation. BLP-tolerized, WT BMDM exhibited a markedly attenuated IRAK-1protein expression (Fig. 6A, 6B), and an impaired ability to formboth TLR2–MyD88 and MyD88–IRAK immunocomplexes (Fig.6C–E) in response to a second BLP stimulation, which is consistent

with our previous findings inBLP-tolerized humanmonocytic THP-1 cells (11). Notably, pretreatment of ST2-deficient BMDM with100 ng/ml BLP for 24 h resulted in a decrease in IRAK-1 proteinexpression, which remained persistently at low levels even afterstimulation with a higher dose of BLP at 1000 ng/ml (Fig. 6F, 6G).Furthermore, BLP-pretreated, ST2-deficient BMDM displayeda substantially reduced immunocomplex formation between eitherTLR2 and MyD88 or MyD88 and IRAK-1 after a second BLPstimulation (Fig. 6H–J), indicating that, like WT monocytes/mac-rophages, induction of BLP tolerance in ST2-deficientmacrophagesis also characterized by reduced IRAK-1 protein expression andattenuated associations of TLR2–MyD88 and MyD88–IRAK.

DiscussionIt is well documented that ST2, a member of the TIR superfamily,exerts an inhibitory effect on Gram-negative LPS-stimulated proin-flammatory cytokine production by attenuation of TLR4 expression(17), IkB degradation (18), and NF-kB binding to the promoter ofproinflammatory cytokine IL-6 (18). Consequently, administrationof a soluble form of ST2 protects mice against endotoxin-inducedshock (17) and ischemia/reperfusion-related lethality (23). Recentwork by Brint et al. (19) further demonstrated that ST2 is a negativeregulator for TLR4 signaling. In the absence of this receptor, ST2-deficient macrophages produced significantly increased amountsof proinflammatory cytokines TNF-a, IL-6, and IL-12 in responseto LPS stimulation. Furthermore, ST2 attenuated TLR4-mediatedNF-kB activation via sequestration of the TLR proximal signaling

FIGURE 3. Inhibition by ST2 in BLP and MyD88, but not IRAK-1, induced NF-kB activation. A, HEK-hTLR2 cells were cotransfected with an NF-kB

gene construct and various amounts of plasmid encoding Flag-tagged ST2, followed by stimulation with 100 ng/ml BLP for 6 h. B and C, HEK-hTLR2 cells

were transfected with an NF-kB gene construct, and cotransfected with plasmids encoding Flag-tagged ST2, MyD88 (B), and IRAK-1 (C). Empty vector

(EV) was used as the control. NF-kB activation was expressed as relative stimulation of luciferase activity (fold stimulation). Data are expressed as the

mean 6 SD of triplicate samples and representative of at least three to five separate experiments.

A B

C D E

FIGURE 2. Enhanced degradation of IRAK-1 protein and increased formation of TLR2–MyD88 and MyD88–IRAK-1 immunocomplexes in BLP-

stimulated, ST2-deficient macrophages. BMDM isolated from WT and ST2-deficient (knock-out [KO]) mice were stimulated with 1000 ng/ml BLP for the

indicated periods. Cytoplasmic proteins were extracted and subjected to immunoblotting (A) or immunoprecipitation (B) as described in the Materials and

Methods. Bands corresponding to signals of either IRAK-1 (C) in the lysate or TLR2 (D) and IRAK-1 (E) in the immunoprecipitation were scanned and

analyzed. The intensity of each band was corrected by its corresponding GAPDH band and expressed as the percentage of the intensity detected in the

unstimulated macrophages. All data are presented as the mean 6 SD of three separate experiments. pp , 0.05 compared with WT macrophages.

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components, MyD88 and Mal (19). Although ST2-deficient mac-rophages produced more IL-12 than did WT macrophages in re-sponse to stimulation with a TLR2 agonist, BLP (19), it is largelyundefined whether ST2 also negatively regulates TLR2 signaling.In the current study, we demonstrated that stimulation of ST2-

deficient macrophages with different TLR2 agonists BLP, LTA, andPGN resulted in a substantially increased production of proin-flammatory cytokines, indicating an inhibitory function of ST2 on theTLR2-mediatedinflammatoryresponse.Thiswasfurthersupportedbythe finding that overexpression of ST2 dose-dependently attenuatedGram-positive BLP stimulated NF-kB activation. To elucidate themolecular mechanisms by which ST2 negatively regulates TLR2signaling, we first examined the effect of ST2 on overexpression ofMyD88 and IRAK-1 induced activation of their downstream signal-ing pathway. Cotransfection of ST2 dose-dependently suppressedMyD88, but not IRAK-1, induced NF-kB activation in HEK-hTLR2cells, suggesting that ST2 achieves its downregulation of TLR2 sig-naling by targeting upstream rather than downstream of IRAK-1.This is consistent with the previous finding in which coexpressionof ST2 inhibited NF-kB activation induced by MyD88, but notIRAK-1 in HEK293 cells stably transfected with type I IL-1 receptor(HEK293RI cells) (19). After this, we further analyzed the influenceof ST2 on BLP-stimulated activation of the TLR2 proximal signal-ing components. Compared with WT macrophages, ST2-deficientmacrophages displayed a substantially enhanced formation of bothTLR2–MyD88 and MyD88–IRAK immunocomplexes upon BLPstimulation, indicating that ST2 possesses an inhibitory action onBLP-stimulated TLR2 signaling by suppressing the recruitment ofTLR2, MyD88, and IRAK-1 to form immunocomplexes.In contrast to our in vitro finding of the dramatically increased

release of proinflammatory cytokines in BLP-stimulated, ST2-

deficient macrophages, serum levels of TNF-a in ST2-deficientmice were identical to those observed in WT mice after a lethalBLP challenge, although ST2-deficient mice exhibited higher se-rum levels of IL-6. Consistent with the similarity of proin-flammatory cytokine production between ST2-deficient and WTmice, ST2-deficient mice did not show any significantly increasedsusceptibility to BLP-induced lethality, with comparable mortalityrates to those observed in BLP-challenged WT mice. This contro-versy after BLP stimulation between ST2-deficient macrophagesin vitro and ST2-deficient mice in vivo was also found in LPS-stimulated ST2-deficient mice by Brint et al. (19), in which ST2-deficient mice displayed an equivalent sensitivity to LPS in vivowhen compared with their WT littermates, with identical mortalityrates and circulating proinflammatory cytokine levels (19). Theseauthors suggested a plausible interpretation for this controversialphenomenon. As ST2 is not constitutively expressed and externalstimuli are required for the expression of ST2, the lag period forST2 induction might be sufficient for the development of LPS-induced shock (19). A recent study byWieland et al. (24) also foundthat ST2-deficient mice displayed an equal susceptibility toMycobacterium tuberculosis infection as WT mice, despite therebeing amodest shift toward the Th1 response in ST2-deficient mice.Tolerance to bacterial cell wall components, including Gram-

negative LPS and Gram-positive BLP, represents an essential reg-ulatory mechanism of the host innate immune system during bac-terial infection (4, 5, 7). LPS tolerance has long been the focus ofextensive scientific and clinical research, in an attempt to identifythe consequence of changes that occur at a molecular level (4, 25).Emerging evidence has revealed that LPS-induced tolerance cor-relates closely with multiple alterations in the TLR4 signalingpathway that involves cell surface molecules, intracellular signal-ing components, proinflammatory and anti-inflammatory gene ex-pression profiles (25–28). More recently, Brint et al. (19) found thatboth ST2-deficient macrophages and mice failed to develop LPStolerance, and they further demonstrated that ST2 functions asa crucial mediator for LPS-induced tolerance by sequestering theadaptor protein MyD88. As acquisition of LPS tolerance is a majorcontributor to an increased secondary bacterial infection in hospi-talized patients (4, 6), targeting ST2 specifically in the future mayhave the therapeutic potential in these patients.Tolerance to theGram-positive bacterial cellwall component, BLP

has been shown to be associated with downregulation of the TLR2-mediated signal transduction pathway (7, 10, 11, 21, 29) and pro-tection against microbial sepsis-related lethality (7–9, 30). However,it is undetermined whether induction of BLP tolerance, like LPS-induced tolerance, is dependent on ST2. We first assessed the re-quirement of ST2 in BLP-induced tolerance in vitro. A moderate but

A B

FIGURE 5. Improved survival and attenuated serum proinflammatory cytokines in BLP-tolerized, ST2-deficient mice exposed to a lethal septic chal-

lenge. A, WT and ST2-deficient mice were injected i.p. with either PBS (naive) or 10 mg/kg BLP (BLP-tolerized), and 24 h later all mice received i.p.

a lethal dose of BLP at 45 mg/kg. Survival was monitored for at least 7 d. B,TNF-a and IL-6 concentrations in the serum, collected at 90 min and 4 h after

mice challenged with 45 mg/kg BLP, were assessed by cytometric bead array. Data are expressed as the mean 6 SD of five mice per time point. pp , 0.05

compared with naive mice; ppp , 0.05 compared with WT mice.

FIGURE 4. Attenuated proinflammatory cytokine release from BLP-

pretreated, ST2-deficient macrophages in response to a second BLP

stimulation. WTand ST2-deficient peritoneal macrophages were pretreated

with various doses of BLP for 24 h, and further stimulated with 1000 ng/ml

BLP for 16 h. TNF-a and IL-6 concentrations in the culture supernatants

were assessed by cytometric bead array. Data are expressed as the mean 6SD of duplicate samples and representative of at least four to six separate

experiments. pp, 0.05 compared with naive macrophages stimulated with

1000 ng/ml BLP.

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substantially diminished production of proinflammatory cytokinesTNF-a and IL-6 in response to a second BLP stimulation was sub-stantiated in BLP-pretreated, ST2-deficient macrophages, demon-strating that BLP tolerance develops in ST2-deficient macrophagesdespite the absence of this receptor. To further validate our in vitrofinding in an in vivo setting, we challenged ST2-deficient mice witha lethal dose of BLP. Pretreatment of ST2-deficient mice witha nonlethal dose of BLP conferred an improved survival advantageagainst BLP-related lethality and attenuated proinflammatory cyto-kine release, which are similar to those observed in BLP-pretreated,WT mice. These data indicate that ST2 is not required for BLP-induced tolerance in vivo. In addition, substantially reduced ex-pression of IRAK-1 protein and attenuated association of TLR2–MyD88 and MyD88–IRAK immunocomplexes, two critical molec-ular events of the TLR2 upstream signaling pathway characterized inthe BLP-tolerized cells (7, 11), were observed in BLP-pretreated,ST2-deficient macrophages after a second BLP stimulation, whichfurther support our notion that BLP-induced tolerance proceedswithout the ST2 receptor. However, the effect of BLP-induced tol-erance, in terms of the attenuated proinflammatory cytokine releaseand improved survival was less pronounced in ST2-deficient mac-rophages and mice than WT ones, implicating that the influence ofST2 on BLP-induced tolerance could not be fully excluded and ST2may play a minor contributory role.The natural ligand of ST2 has been recently identified as IL-33,

a newmember of the IL-1 family (31). IL-33 functions as a cytokineand signals through its interaction with a receptor complex con-

sisting of ST2 and IL-1 receptor accessory protein (31–34). A re-cent study by Carriere et al. (35) further discovered that IL-33 is thesame molecule as the NF from high endothelial venules that en-dows with transcriptional repressor characteristics (36), indicatingIL-33 may have intracellular functions that are independent ofbinding to the ST2 receptor. Thus, IL-33, like IL-1a and highmobility group box protein-1, may possess dual functions by actingas either a proinflammatory cytokine or a transcription factor (37).As the IL-33 receptor ST2 has been demonstrated to be a crucialmediator for LPS-induced tolerance (19), it is reasonable to spec-ulate that stimulation of ST2 by its functional ligand IL-33 couldlead to a tolerance state to LPS. However, Espinassous et al. (38)recently reported that IL-33 does not induce LPS desensitization inmurine macrophages, but instead increases LPS-stimulated proin-flammatory cytokine production by upregulating the LPS receptorcomplex TLR4–MD2 and the adaptor protein MyD88.Takentogether, in thecurrentstudywehavedemonstrated thatST2

negatively regulatesTLR2signaling,but incontrast to the roleofST2in mediating LPS-induced tolerance, BLP-induced tolerancedevelops in the absence of ST2. Despite the fact that both TLR2 andTLR4 signal via the same adaptor protein, MyD88, there are sig-nificant differences in reprogramming of the intracellular signaltransduction pathway and antimicrobial activity between TLR2-mediated tolerance (e.g., BLP tolerance) and TLR4-mediated tol-erance (e.g., LPS tolerance) (7). The different involvements of ST2in LPS-induced tolerance and BLP-induced tolerance further sup-port this notion.

FIGURE 6. AttenuatedTLR2-mediated

signal transduction pathway in BLP-

tolerized, ST2-deficient macrophages. WT

(A, B) and ST2-deficient (KO; C, D)

BMDM were pretreated with culture me-

dium (naive) or 100 ng/ml BLP (BLP-

tolerized) for 24 h, and further stimulated

with 1000 ng/ml BLP for the indicated

periods. Cytoplasmic proteins were ex-

tracted and subjected to immunoblotting

(A, F) or immunoprecipitation (C, H) as

described in the Materials and Methods.

Bands corresponding to signals of either

IRAK-1 (B,G) in the lysate or TLR2 (D, I)

and IRAK-1 (E, J) in the immunoprecipi-

tation were scanned and analyzed. The

intensity of each band was corrected by its

corresponding GAPDH band and ex-

pressed as the percentage of the intensity

detected in the unstimulated, naive mac-

rophages. All data are presented as the

mean6 SD of three separate experiments.

pp , 0.05 compared with naive macro-

phages. KO, knock-out.

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DisclosuresThe authors have no financial conflicts of interest.

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