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Differential Activation of Human TLR4 by and Shigella flexneri 2a : Combined Effects of A Acylation State and TLR4 This information is current as Polymorphisms on Signaling of October 5, 2021. Prasad Rallabhandi, Agnes Awomoyi, Karen E. Thomas, Armelle Phalipon, Yukari Fujimoto, Koichi Fukase, Shoichi Kusumoto, Nilofer Qureshi, Marcelo B. Sztein and Stefanie N. Vogel Downloaded from J Immunol 2008; 180:1139-1147; ; doi: 10.4049/jimmunol.180.2.1139 http://www.jimmunol.org/content/180/2/1139 http://www.jimmunol.org/ References This article cites 46 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/180/2/1139.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Activation of Human TLR4 by Escherichia coli and Shigella flexneri 2a Lipopolysaccharide: Combined Effects of Acylation State and TLR4 Polymorphisms on Signaling1

Prasad Rallabhandi,2* Agnes Awomoyi,2* Karen E. Thomas,* Armelle Phalipon,‡ Yukari Fujimoto,§ Koichi Fukase,§ Shoichi Kusumoto,§ Nilofer Qureshi,¶ Marcelo B. Sztein,3† and Stefanie N. Vogel3,4*

The lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been Downloaded from associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling. HEK293T transfectants expressing wild-type or polymor- phic TLR4 were stimulated with Escherichia coli (predominantly hexaacylated lipid A) or Shigella flexneri 2a (a mixture of hexaacylated, pentaacylated, and predominantly tetraacylated lipid A) LPS, or hexaacylated vs pentaacylated synthetic lipid As. NF-␬B-reporter activity was significantly lower in response to S. flexneri 2a than E. coli LPS and further decreased in polymorphic transfectants. Neither hexaacylated nor pentaacylated synthetic lipid A induced NF-␬B activity in wild-type transfectants under http://www.jimmunol.org/ the identical transfection conditions used for LPS; however, increasing human MD-2 expression rescued responsiveness to hexaacylated lipid A only, while murine MD-2 was required to elicit a response to pentaacylated lipid A. Adherent PBMC of healthy volunteers were also compared for LPS-induced TNF-␣, IL-6, IL-1␤, and IL-10 production. levels were signif- icantly lower (ϳ20–90%) in response to S. flexneri than to E. coli LPS/lipid A and PBMC from polymorphic individuals secreted decreased cytokine levels in response to both LPS types and failed to respond to pentaacylated lipid A. Thus, the combination of acylation state and host genetics may significantly impact vaccine immunogenicity and/or efficacy, whether LPS is an integral component of a whole organism vaccine or included as an adjuvant. The Journal of Immunology, 2008, 180: 1139–1147.

n response to infection, the capacity of the host to mount an side chain (which confers serologic specificity to the organism), a by guest on October 5, 2021 effective, early innate is a key determinant more conserved “core” oligosaccharide that, in turn, is covalently I of disease resistance or susceptibility. The outer membrane linked to a hydrophobic portion, lipid A. Lipid A is of Gram-negative is comprised predominantly of endo- responsible for the biological activity of LPS and has been termed toxic LPS. LPS elicits a wide variety of biological activities in the the “endotoxic principle” of LPS (4). Proinflammatory signal ini- host (1). For most LPS species, TLR4 acts as the “pattern recog- tiation by LPS depends upon the interaction of the TLR4 nition receptor” that senses LPS, and initiates intracellular signal- complex (i.e., TLR4, MD-2, and CD14) with the lipid A moiety of ing leading to an inflammatory response (2, 3). LPS is comprised LPS. Structural variations within lipid A, such as the number and of three covalently linked regions: a repeating O- positions of acyl chains or the number and position of charged groups on the backbone of the lipid A , greatly influence this interaction (5). Using infrared attenuated total re- *Department of and Immunology and †Center for Vaccine Develop- ment, University of Maryland, Baltimore, MD 21201; ‡Institute Pasteur, Paris, flectance spectroscopy, Seydel et al. (6) showed that the endotoxic France; §Osaka University, Osaka, Japan; and ¶Department of Basic Medical Science, effect of lipid A portion of LPS directly correlated with, and School of Medicine, University of Missouri, Kansas City, MO 64108 strongly depends upon, the tilt angle of the sugar backbone with Received for publication March 7, 2007. Accepted for publication November respect to the membrane surface, and the number and distribution 12, 2007. of the hydrocarbon chains. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance It also has been shown that only those LPS comprised with 18 U.S.C. Section 1734 solely to indicate this fact. of a hexaacylated lipid A moiety with a conical/concave shape are 1 This work was supported, in part, with federal funds from National Institute of highly active biologically (5, 6). In contrast, lipid A molecules that and Infectious Diseases, National Institutes of Health, Department of Health are either tetraacylated or pentaacylated exhibit a cylindrical mo- and Human Services, under Contract No. N01-AI-30028 (to M.B.S.) and Grants AI057927 (to M.B.S.), AI18797 (to S.N.V.), and GM50870 (to N.Q.). lecular shape and greatly attenuated endotoxic activity, and may 2 P.R. and A.A. contributed equally to this study. even exhibit antagonistic activity (6, 7). For example, Hajjar et al. 3 M.B.S. and S.N.V. contributed equally to the direction of this work. (8) found that a hexaacylated Pseudomonas aeruginosa LPS was much more stimulatory in cells expressing human TLR4 than pen- 4 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, University of Maryland, 660 West Redwood taacylated LPS prepared from a laboratory . Therefore, in Street, Room 324, Baltimore, MD 21201. E-mail address: svogel@som. response to a Gram-negative bacterial infection or vaccine, or in umaryland.edu response to a vaccine in which a TLR4 agonist is present as an Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 adjuvant, the relative contribution of TLR4-mediated signaling to www.jimmunol.org 1140 LPS ACYLATION STATE AND TLR4 SNPs ALTER LPS RESPONSIVENESS

the initial host innate immune response may differ depending on their capacity to elicit TLR4-mediated responses, first in a tran- the acylation state of the TLR4 agonist. The induction of a proin- sient transfection system in HEK293T cells (3), and then in flammatory cytokine milieu and up-regulation of costimulatory human PBMC derived from WT (nonpolymorphic) and poly- molecules induced by LPS activation of TLR4 impacts greatly on morphic individuals. The rationale for using Shigella flexneri 2a the subsequent acquired immune response. In this context, struc- was based on the facts that 1) S. flexneri 2a is the single most tural variations such as the degree of acylation within the lipid A important S. flexneri subserotype that causes shigellosis in de- moiety could contribute significantly toward determining the im- veloping countries, and 2) attenuated S. flexneri 2a vaccines are munogenicity and/or efficacy of a specific Gram-negative vaccine. at an advanced stage of development and currently being eval- It has been previously shown that the Escherichia coli LPS is uated in clinical trials (15). In contrast to a previous report (9), comprised mainly of hexaacylated lipid A, whereas Shigella flex- we demonstrate that the S. flexneri 2a LPS used in this study is neri LPS has been reported to be predominantly pentaacylated (9). comprised of a mixture of hexaacylated, pentaacylated, and pre- Therefore, it would be anticipated that the overall TLR4 response dominantly tetraacylated lipid A-containing LPS species. The to S. flexneri 2a LPS would be less than that of cells stimulated lipid A acylation state for the S. flexneri 2a strain used in this with E. coli LPS. study differs from the WT S. flexneri serotype 5a that expressed Just as there are variations in the microbial structures that mainly hexaacylated lipid A-containing LPS species, as re- contribute to the host response, the innate immune response is ported by D’Hauteville et al. (16). Consistent with previous also subject to genetic regulation. TLR4 is a highly conserved observations, S. flexneri 2a LPS elicited significantly dimin- transmembrane receptor for LPS. Arbour et al. (10) identified ished TLR4-mediated signaling when compared with E. coli two TLR4 single nucleotide polymorphisms (SNPs)5 that en- LPS (shown to possess predominantly hexaacylated lipid A- code single amino acid substitutions, Asp299Gly (D299G) and containing LPS), and the response to both LPS preparations was Downloaded from Thr399Ile (T399I), in the ectodomain of human TLR4. These further diminished in cells expressing the TLR4 polymorphic two polymorphisms were identified in ϳ6 and ϳ3% of individ- variants. Interestingly, the response to synthetic hexaacylated uals, respectively, and may be expressed concurrently (10). In- lipid A by WT transfectants required much higher human MD-2 heritance of these SNPs, singly or together, was associated with expression than for E. coli LPS, yet no response to pentaacy- an LPS-hyporesponsive phenotype in human airway epithelial lated lipid A was detected unless rescued by murine MD-2. cells and alveolar , and both SNPs were associated Taken collectively, these data support the hypothesis that both http://www.jimmunol.org/ with a significantly blunted response to inhaled LPS. Kiechl the structural makeup of the LPS, in concert with inherited et al. (11) reported that subjects who expressed the Asp299Gly structural modifications to TLR4, can combine to affect signif- SNP were more susceptible to Gram-negative bacterial infec- icantly the outcome of the host response to infection by Gram- tions, and Lorenz et al. (12) reported that expression of the negative organisms or in vaccines that contain intrinsic or ex- TLR4 Asp299Gly variant was associated with septic trinsic TLR4 agonists in a significant number of individuals. patients, and that patients with Asp299Gly or Thr399Ile forms exhibited a higher incidence of Gram-negative infection. Collectively, these data support the hypothesis that Materials and Methods by guest on October 5, 2021 faulty TLR4 signaling fails to induce a sufficiently robust in- Cells and reagents flammatory response to control infection. HEK293T human embryonic kidney cells (American Type Culture Col- We recently reported that expression of TLR4 molecules that lection) were cultured in DMEM (BioWhittaker) supplemented with 10% ␮ carry the Asp299Gly and/or Thr399Ile extracellular polymorphisms FBS, 2 mM L-glutamine, 100 U/ml , and 100 g/ml streptomycin. Protein-free E. coli K235 LPS (17) and S. flexneri 2a (18, 19) were used in in HEK293T cells results in mitigated host responses to three this study and synthetic hexaacylated lipid A and pentaacylated lipid A structurally unrelated TLR4 agonists: Gram-negative LPS, the fu- were synthesized as described elsewhere (20). The hexaacylated synthetic sion (F) protein of respiratory syncytial virus, and Chlamydia heat lipid A used in this study (compound 506, LA-15-PP) has the following shock protein 60 (3). In all cases, the pattern of responsiveness to structure: ␤(1–6) bisphosphorylated at the 1 and 4Ј positions with the acylated pattern of 3Ј position 14:0 [3-O (14:0)], 2Ј these three agonists was similar: transfectants expressing either the 299 399 position 14:0 [3-O (12:0)], 3 position 14:0 (3-OH), and 2 position 14:0 Asp Gly or Thr Ile were less responsive than cells expressing (3-OH) (21). The synthetic pentaacylated lipid A used in this study, LA- wild-type (WT) TLR4, but more responsive than HEK293T cells 21-PP, has the following structure: 3Ј position 14:0 (3-OH), 2Ј position expressing the doubly mutated TLR4 vector (i.e., both SNPs are 14:0 [3-O (16:0)], 3 position 14:0 (3-OH), 2 position 14:0 (3-OH). The expressed on the same chromosomal homolog). Molecular mod- biological activity of this synthetic pentaacylated lipid A was described previously by Rietschel et al. (22). SuperFect transfection reagent was eling revealed that the amino acids located in positions 299 and obtained from Qiagen. All expression constructs were prepared 399 of TLR4 lie on the same extracellular “face” of the TLR4 using the EndoFree Plasmid Maxi kit (Qiagen). protein and that expression of the polymorphic amino acids might create steric and charge alterations that could potentially Plasmid constructs alter its interaction with ligand or common coreceptor mole- The expression vector, pcDNA3-TLR4, that encodes an untagged WT cules (3, 13, 14). TLR4 was provided by Dr. E. Lorenz (University of North Carolina, In this study, we sought to extend our previous findings by Chapel Hill, NC). Point mutations encoding an aspartic acid (Asp) to gly- analyzing the capacity of two species of LPS or synthetic lipid cine (Gly) substitution at the amino acid position 299 (D299G) and a thre- A that differ in the degree of acylation to modulate the host onine (Thr) to isoleucine (Ile) substitution at amino acid 399 (T399I) in innate immune response in cells that express either WT or poly- human TLR4 protein were created by site-directed mutagenesis using a QuikChange mutagenesis kit (Stratagene) according to the manufacturer’s morphic TLRs. To this end, we tested highly purified LPS prep- instructions as described elsewhere (3). arations derived from E. coli and S. flexneri 2a, as well as syn- Expression , pcDNA3-huCD14 and pEFBOS-HA-huMD-2, the thetic hexaacylated and pentaacylated lipid A molecules, for ELAM-1 luciferase, pELAM-luc (NF-␬B reporter), and pCMV1-␤-galac- tosidase reporter plasmids were also provided by Dr. D. Golenbock (Uni- versity of Massachusetts Medical School, Worcester, MA) and have been 5 Abbreviations used in this paper: SNP, single nucleotide polymorphism; WT, wild described elsewhere (3, 23, 24). The murine MD-2 construct, pEFBOS- type; hu, human; mu, murine; RLU, relative luciferase unit; HEK, human embryonic muMD-2, was provided by Dr. K. Miyake (University of Tokyo, Tokyo, kidney; MS, mass spectroscopy; MD-2, myeloid differentiation protein-2. Japan) and has been described elsewhere (25). The Journal of Immunology 1141

Mass spectrometry of lipid A species and stored at Ϫ80°C until analyzed for (A–H) by cytometric bead array (CBA; BD Biosciences). MALDI-TOF mass spectra of the purified were acquired in the pos- itive- and negative-linear mode using an AXIMA-CFR mass spectrometer Cytokine bead from Kratos Analytical. The AXIMA-CFR is equipped with a 337-nm nitrogen laser, a 20-kV extraction voltage, and time-delayed extraction. The CBA is a multiplex, particle-based immunoassay that uses the sensi- Saturated 6-aza-2-thiothymine in 50% acetonitrile and 10% tribasic am- tivity of amplified fluorescence detection by flow cytometry to measure monium citrate (9:1, v/v) served as the matrix. Purified monophosphoryl soluble analytes. Four soluble analytes (IL-1␤, IL-6, IL-10, and TNF-␣) lipid A was prepared by hydrolyzing the LPS in 0.1 N HCl at 100°C for 15 were measured using a Beckman Coulter Epics Elite ESP flow cytometer min. The lipid A was extracted as described previously (26, 27). The lipid and simple linear regression to determine absolute concentrations using the A samples dissolved in chloroform/methanol (4:1, v/v) were deposited onto CBA Excel software (BD Biosciences). The limit of detection for the the sample plate with an equal portion of the matrix solution (0.8 ␮l). The above-mentioned cytokines was 10 pg/ml. sample mixtures were dried to room temperature before mass analysis. hexaacylated lipid A 1,4Ј-bisphosphate from WT E. coli (Sigma-Aldrich) Statistical analyses served as an external standard for calibration. Distribution of TLR4 genotypes and alleles were determined by simple Reporter assay gene counting. Quantitative data of NF-␬B activation in transfected cells are presented as mean Ϯ SE and were analyzed using a one-way ANOVA HEK293T cells were seeded into 12-well Costar plates (Corning) at a den- with repeated measures, followed by post-hoc comparisons using Tukey’s ϫ 5 sity of 2 10 cells/well and incubated overnight in a CO2 incubator. The multiple paired comparison test (GraphPad PRISM 4 program for Win- next morning, cells were cotransfected for 3 h with the optimized amounts dows). Nonparametric analysis of cytokine responses of human mononu- of pcDNA3-TLR4, pcDNA3-human (hu) CD14, and pEFBOS-HA- clear cell cultures was performed using the Wilcoxon-sign ranked sum test huMD-2 (3) together with the NF-␬B reporter (500 ng/well) and pCMV1- for comparison of cytokines produced in response to E. coli vs S. flexneri ␤Gal (100 ng/well). The final DNA concentration was adjusted to 1.5 ␮g/ 2a LPS in individual subjects (regardless of their TLR4 genotype) whereas Downloaded from well with the pcDNA3 blank vector (Invitrogen Life Technologies). The the Mann-Whitney ranked sum test was used to determine whether there transfection was conducted with SuperFect transfection reagent (Qiagen). were significant differences in cytokines elicited between TLR4 polymor- Cells were allowed to recover for 20 h, washed with 1ϫ PBS, and stim- phic vs nonpolymorphic PBMC to either E. coli or S. flexneri 2a LPS ulated with E. coli or S. flexneri 2a LPS or hexaacylated or pentaacylated preparations (SigmaStat for Windows version 3.1; Systat Software). lipid A at the indicated concentrations for 5 h. Cells were lysed in 1ϫ reporter assay buffer (Promega), and ␤-galactosidase (Galacto-Light system; Tropix) and luciferase (luciferase assay system; Promega) activi- Results ties were analyzed, using a Berthold LB 9507 Luminometer (Berthold Chemical analysis of lipid A derived from E. coli K235 http://www.jimmunol.org/ Technologies). “Relative luciferase activity” was calculated by normalizing and S. flexneri 2a LPS each sample’s luciferase activity for constitutive ␤-galactosidase activity mea- sured within the same sample, and represented as relative luciferase units The E. coli K235 and S. flexneri 2a LPS preparations used for this (RLU) as described previously (3). study were subjected to lipid A analysis by thin layer chromatog- raphy and mass spectroscopy (MS). Both thin layer chromatogra- Murine cultures phy (data not shown) and MS revealed that while the lipid A from Peritoneal exudate macrophages were obtained from 5- to 6-wk-old E. coli was predominantly hexaacylated (Fig. 1A), with lesser C57BL/6J mice (The Jackson Laboratory) by lavage 5 days after i.p. in- amounts of pentaacylated and tetraacylated species, the lipid A jection of 3 ml of sterile 3% thioglycolate broth (28). Cells were washed from S. flexneri 2a was a mixture comprised predominantly of and resuspended in RPMI 1640 containing 2% FCS and standard supple- by guest on October 5, 2021 ments. Macrophages were plated in 24-well tissue-culture dishes (2 ϫ 106 tetraacylated species, with some pentaacylated lipid A and a small cells/well) for supernatant collection. After overnight incubation to allow amount of hexaacylated lipid A (Fig. 1B). Although these results for adherence of macrophages, monolayers were washed to remove non- are consistent with those previously reported for the E. coli lipid A adherent cells and incubated with medium, LPS, or lipid A at the indicated structure (9), they differ from a previous report that determined the concentrations in a final volume of 1 ml. Supernatants were harvested 24 h lipid A structure of another S. flexneri (strain not designated) to be after treatment and stored at Ϫ70°C. Murine IL-␤, IFN-␥, RANTES, and TNF-␣ were detected using the Ab pairs and standards provided in the predominantly pentaacylated (9). Quantikine M ELISA kit (R&D Systems). All experiments using murine macrophages were conducted with institutional approval. S. flexneri 2a LPS is less potent than E. coli LPS in TLR4-mediated activation of NF-␬B-luciferase reporter activity Subjects, isolation of PBMC, and genotyping of TLR4 Asp299Gly and Thr399Ile polymorphisms To compare the relative capacity of LPS derived from E. coli vs. S. flexneri 2a to activate human TLR4, a transient transfection Seventy-three healthy male and female volunteers, between 20 and 60 system in which HEK293T cells were transfected with human years of age, were recruited from the Baltimore-Washington area and Uni- versity of Maryland campus (Baltimore, MD; UMB) to participate in this TLR4/MD-2/CD14 vectors, along with NF-␬B luciferase and study. The purpose of the study was explained to the volunteers and they ␤-galactosidase reporter constructs, was initially used. Transfec- signed informed consent forms before blood donations. Study protocols tions were conducted using concentrations of expression con- were approved by the UMB Internal Review Board. PBMC were isolated structs previously optimized for the measurement of TLR4-medi- by density gradient centrifugation and cryopreserved in liquid N as pre- 2 ␬ viously described (29). DNA was extracted from PBMC according to a ated NF- B signaling in response to the identical preparation of E. standard procedure (Puregene DNA Purification System). DNA samples coli K235 LPS used in this study (3). Because the HEK293T cells were genotyped using a fluorogenic 5Ј nuclease TaqMan assay in the ABI do not have endogenous TLR4, they respond to LPS only when PRISM 7900HT Sequence Detection System (Applied Biosystems) (30). transfected with the TLR4 complex (3). In multiple, side-by-side Thereafter, TaqMan-based results were validated for every DNA sample by experiments, both E. coli LPS and S. flexneri 2a LPS induced sequencing (Biopolymer Core Facility, UMB) after labeling DNA with the Big Dye Terminator Cycle Sequencing kit, version 3.1 (Applied Biosys- dose-dependent NF-␬B-driven luciferase activity; however, the S. tems) as previously described (24). flexneri 2a LPS was consistently less stimulatory when compared with the E. coli LPS at the same LPS concentrations ( p Ͻ 0.05) PBMC cultures (Fig. 2). These differences confirm and extend previous observa- PBMC prepared from 8 TLR4 polymorphic and 22 nonpolymorphic vol- tions that predominantly hexaacylated, lipid A-containing LPS 6 unteers were seeded at 2 ϫ 10 cells into 24-well Costar tissue-culture (e.g., E. coli) preparations are more stimulatory than LPS prep- plates. Cells were allowed to adhere for5hat37°C in 5% CO2. Nonad- herent cells were removed by thorough washing with Hanks’ buffer. Ad- arations that contain predominantly hypoacylated (e.g., penta- herent cells (predominantly ) were stimulated for 18 h with acylated and tetraacylated) lipid A species as contained in the S. medium alone, LPS, or lipid A. Culture supernatants were harvested flexneri 2a LPS (5, 6, 8). 1142 LPS ACYLATION STATE AND TLR4 SNPs ALTER LPS RESPONSIVENESS

FIGURE 1. Lipid A analysis of E. coli and S. flexneri 2a LPS preparations used in this study. Monophospho- ryl lipid A prepared from the LPS of E. coli K235 and S. flexneri 2a was analyzed by mass spectrometry (A and B, respectively) as described in Materials and Methods. Analysis of the two LPS preparations by MS revealed that, whereas the lipid A from E. coli LPS was predom- inantly hexaacylated (A), the lipid A from S. flexneri 2a LPS was predominantly tetraacylated (where the myr- istoxymyristate was missing from the 3Ј position of the glucosamine disaccharide), pentaacylated (the hydroxy- myristate was missing from the 3-position), and hexaacylated forms were also present (B). This analysis was conducted on two separate preparations of S. flex- neri 2a with similar results. Downloaded from

TLR4 signaling induced by hexaacylated lipid A, but not were titrated in our previous study to elicit optimal E. coli LPS- pentaacylated lipid A, requires higher expression levels of induced activation (3). However, under these identical transfection human MD-2 in HEK293T transfectants conditions where both E. coli and S. flexneri 2a LPS elicited robust ␬ To explore in greater detail the effects of acylation differences in NF- B-driven luciferase activity (Fig. 2), neither synthetic http://www.jimmunol.org/ lipid A on TLR4 signaling, we next compared the response of hexaacylated nor pentaacylated lipid A preparations induced any HEK293T cells transfected with the TLR4-signaling complex to measurable activity, even at a final lipid A concentration of 8 stimulation by synthetic lipid A preparations that were either ␮g/ml (Fig. 3A). Based on our previous observations in murine hexaacylated or pentaacylated. It has been previously reported that macrophages that significantly higher concentrations of E. coli cylindrically shaped tetraacylated and pentaacylated lipid A lipid A were required to drive cytokine secretion than E. coli LPS species are less biologically active than conical/concave-shaped (31), and the observations of others that MD-2 greatly augments hexaacylated lipid A (5–7). Therefore, we reasoned that the penta- LPS signaling by TLR4 (32), we hypothesized that, perhaps, acylated and hexaacylated species of lipid A that are present in the higher levels of human MD-2, in addition to higher concentrations

S. flexneri 2a LPS, and not the tetraacylated species, are primarily of lipid A, might be necessary for lipid A to induce activity in this by guest on October 5, 2021 responsible for its observed biological activity. Therefore, in the system. This was indeed the case for the hexaacylated lipid A. next series of experiments, we compared pentaacylated and When the input human MD-2 expression vector concentration was hexaacylated lipid A preparations to study the MD-2 requirement increased 100-fold to 300 ng/well (Fig. 3B), we observed robust for biological activity. Initially, we stimulated HEK293T cells that activation for the hexaacylated lipid A. In contrast, synthetic pen- had been transfected with expression vectors that encode WT hu- taacylated lipid A still failed to elicit any NF-␬B activation, even man TLR4 (300 ng/well), huCD14 (30 ng/well), and huMD-2 (3 when the MD-2 vector input concentration was increased to 600 ng/well), along with the NF-␬B reporter construct, each of which ng/well (data not shown). This result clearly underscores the im- portance of structural modifications, e.g., the acylation state of the lipid A component of LPS in activating the TLR4-mediated NF- ␬B-driven gene activation, and the important role of MD-2 in this process. It has been previously reported that neither tetraacylated (deacy-

lated) LPS (33) nor lipid IVA (8, 34) are stimulatory in human cells, and in fact, act as antagonists of LPS-mediated signaling. In contrast, in murine cells, tetraacylated LPS/lipid A exhibit agonist activity. To determine whether the decreased activity of the pen- taacylated lipid A that we observed in the HEK293T transfectants was also species-specific, we measured cytokine release in cultures FIGURE 2. S. flexneri 2a LPS shows less activity than E. coli K235 LPS of murine peritoneal macrophages after stimulation with these in the TLR4-mediated activation of NF-␬B. A total of 2 ϫ 105 cells/well same hexaacylated or pentaacylated synthetic lipid A preparations. HEK293T cells were cotransfected with untagged wild-type TLR4 (300 E. coli LPS and S. flexneri 2a LPS were also included as controls. ng/well), huCD14 (30 ng/well) and huMD-2 (3 ng/well), along with the As shown in Table I, the pentaacylated lipid A was active in mu- reporter constructs to obtain optimal E. coli LPS-induced activation. After rine macrophages and induced secretion of IL-1␤, TNF-␣, IFN-␥, overnight recovery, transfected cells were treated with the indicated con- and RANTES to levels similar to those induced by E. coli LPS. centrations of either E. coli K235 LPS or S. flexneri 2a LPS for 5 h. Cells Next, we conducted dose response experiments to determine were washed twice with cold 1ϫ PBS, lysed in 1ϫ lysis buffer, and cen- trifuged to remove cell debris. Luciferase and ␤-galactosidase activities whether a difference between the activity of hexaacylated lipid A were measured in cell lysates as described in Materials and Methods.A and pentaacylated lipid A with respect to their ability to induce p Ͻ 0.05 vs E. coli LPS at cytokine secretion in murine macrophages might be detected at a ,ء .(representative experiment is shown (n ϭ 6 the same concentration. lower concentration than that used in Table I. Table II shows that The Journal of Immunology 1143

Table II. Secretion of IL-1␤ by murine macrophages stimulated with various concentrations of hexaacylated and pentaacylated lipid Aa

Concentration (ng/ml)

0 0.1 1 10 100 1000

Hexaacylated Lipid A Յ7.8b Յ7.8 Յ7.8 18.3 33.5 63.0 Pentaacylated Lipid A Յ7.8 Յ7.8 Յ7.8 23.0 36.1 80.4

a Murine peritoneal macrophages were plated at a density of 1 ϫ 106 cells/ml in a 24-well plate; they were allowed to adhere overnight. Macrophage cultures were then treated for 24 h with media or lipid A and supernatants were assayed for IL-1␤ by ELISA. b Picograms per milliliter.

a minimal concentration of 10 ng/ml is required for either lipid A preparation and that there was no appreciable difference in levels of IL-1␤ released between the two lipid A preparations. These results, taken together, suggest that murine macrophages respond to synthetic pentaacylated lipid A, in contrast to human HEK293T TLR4/MD-2/CD14 transfectants that do not. Downloaded from Murine MD-2 has previously been shown to impart species specificity to another TLR4 agonist, taxol (35), and to rescue

responsiveness to lipid IVA (8, 34). To explore the possibility that the species specificity of pentaacylated lipid A activation in HEK293T cells was due to the species of MD-2, HEK293T cells were transfected with mouse or human MD-2 expression http://www.jimmunol.org/ constructs, along with huTLR4, huCD14, and reporter con- structs, and then treated with medium only, LPS, or lipid A for 6 h. Although LPS preparations and hexaacylated lipid A showed TLR4 activation regardless of the MD-2 species, pen- taacylated lipid A showed TLR4 activation only in the presence of mouse MD-2 (Fig. 3C).

S. flexneri 2a LPS induces diminished TLR4 signaling in

299 399 by guest on October 5, 2021 FIGURE 3. Signaling induced by hexaacylated lipid A through TLR4 HEK293T cells expressing Asp Gly and Thr Ile requires higher expression levels of the coreceptor, MD-2. A total of polymorphic TLR4 variants ϫ 5 2 10 cells/well HEK293T cells were cotransfected with untagged We and others have previously observed that E. coli LPS elicits wild-type TLR4 (300 ng/well), huCD14 (30 ng/well), and the indicated significantly lower responses in transfectants that express amounts of: A, huMD-2 (3 ng/well); B, huMD-2 (300 ng/well) along Asp299Gly and/or Thr399Ile polymorphisms, and that this is most with the reporter constructs. After overnight recovery, transfected cells were treated with the indicated concentrations of E. coli K235 striking in cells that express both polymorphisms (i.e., the TLR4 LPS or S. flexneri 2a LPS or hexaacylated or pentaacylated lipid A for molecules express both SNPs) (3, 10). Therefore, we next sought 299 399 5 h. Luciferase and ␤-galactosidase activities were measured in cell to determine whether the cosegregating Asp Gly and Thr Ile lysates as described in Materials and Methods. A representative exper- polymorphic variant of TLR4 would exhibit a further diminution iment is shown (n ϭ 5). C, Murine MD-2 rescues pentaacylated sig- of responsiveness to the S. flexneri 2a LPS preparations when com- naling by human TLR4. Experiments were set up as described in A and pared with WT TLR4 as observed for E. coli LPS (3). Indeed, B using either human or murine MD-2 (100 ng/well) in conjunction HEK293T cells transfected with the vector that expresses both with human TLR4 and CD14 constructs. A representative experiment is SNPs were consistently and significantly less responsive to both S. ϭ shown (n 3). flexneri 2a as well as E. coli LPS when compared with the WT TLR4 transfectants ( p Ͻ 0.01; Fig. 4).

Table I. Cytokine release by murine macrophage stimulated with E. coli or S. flexneri 2a LPS or synthetic lipid A speciesa

Treatment

Hexaacylated Pentaacylated E. coli S. flexneri 2a Cytokine Media Lipid A Lipid A LPS LPS

IL-1␤ Յ7.8b 66 Ϯ 27 43 Ϯ 927Ϯ 145Ϯ 28 TNF-␣ Յ23.8 20,747 Ϯ 5,586 17,855 Ϯ 389 15,405 Ϯ 1,945 19,022 Ϯ 2,298 IFN-␥ Յ9.4 41 Ϯ 12 30 Ϯ 038Ϯ 13 55 Ϯ 25 RANTES 473.8 Ϯ 5 44,309 Ϯ 5,615 39,529.4 Ϯ 562 42,000 Ϯ 5,095 42,369 Ϯ 2,745

a Murine peritoneal macrophages were plated at a density of 1 ϫ 106 cells/ml and were allowed to adhere overnight. They were treated for 24 h with media, 100 ng/ml LPS, or 1 ␮g/ml lipid A and supernatants were harvested for cytokine production detected by ELISA as described in Materials and Methods. b Picograms per milliliter. 1144 LPS ACYLATION STATE AND TLR4 SNPs ALTER LPS RESPONSIVENESS

FIGURE 4. HEK293T cells expressing Asp299Gly and Thr399Ile poly- morphic TLR4 variant showed diminished response to S. flexneri 2a LPS. A total of 2 ϫ 105 cells/well HEK293T cells were cotransfected with untagged wild-type TLR4 (300 ng/well) or untagged Asp299Gly and Thr399Ile polymorphic TLR4 expression construct (300 ng/well), huCD14 (30 ng/well), and huMD-2 (3 ng/well) along with the reporter constructs. After overnight recovery, transfected cells were treated with the indicated concentrations of E. coli K235 LPS or S. flexneri 2a LPS for 5 h and cell lysates were made. Luciferase and ␤-galactosidase activities were mea- Downloaded from sured in cell lysates as described in Materials and Methods. A represen- tative experiment is shown (n ϭ 5).

Distribution of TLR4 polymorphisms in a normal healthy population http://www.jimmunol.org/ PBMC were purified from blood derived from 73 healthy volun- teers recruited from the greater Baltimore area. Genotyping re- vealed that only 8 individuals were heterozygous for one or both of the two TLR4 polymorphisms (Table III). Seven individuals were heterozygous for the Asp299Gly variant (carrier frequency: 9.6%; minor allele frequency: 4.8%), whereas 4 were heterozygous for 399 the Thr Ile polymorphism (carrier frequency: 5.5%; minor allele FIGURE 5. Cytokine production by PBMC derived from nonpolymor-

frequency 2.7%). Three individuals were doubly heterozygous for phic and TLR4 polymorphic volunteers stimulated with E. coli or S. flex- by guest on October 5, 2021 299 399 both Asp Gly and Thr Ile polymorphisms, 1 was heterozygous neri 2a LPS. Mononuclear cells from 22 wild-type and 8 TLR4 polymor- for only Thr399Ile, and four were heterozygous for Asp299Gly phic individuals were stimulated with 0.05, 0.5, or 5 ng/ml E. coli or S. only. None were homozygous for the minor allele. These data are flexneri 2a LPS as indicated and supernatants collected for cytokine deter- very consistent with many previous reports of allelic frequency in minations (A–H) by cytometric bead array assays as described in Materials healthy populations (12, 30, 36–38). and Methods. Results are shown as mean picograms per milliliter Ϯ SE for all selected volunteers in each group. Numbers above bars represent the Induction of inflammatory cytokines by E. coli vs S. flexneri percent of suppression of cytokine production by cells from polymorphic 2a LPS preparations subjects compared with the corresponding levels observed in supernatants from wild-type (nonpolymorphic) individuals, matched by LPS type and Thus far, all of the comparisons of LPS responsiveness were con- concentration. ducted in the HEK293T transient transfection system. To confirm our findings under more physiological conditions, adherent mono- cytic cell cultures derived from PBMC from 22 WT or 8 poly- pared with those elicited by S. flexneri LPS (Fig. 5, B, D, F, and morphic donors were stimulated with different concentrations of E. H). This trend was observed whether the supernatants were derived coli LPS or S. flexneri 2a LPS. Analysis of cytokine production from WT (nonpolymorphic) or polymorphic individuals. For ex- revealed that at the same concentrations, E. coli LPS (Fig. 5, A, C, ample, at the 5 ng/ml LPS concentration, TNF-␣ production in E, and G) induced higher levels of inflammatory cytokines com- response to S. flexneri LPS was 72.8 and 65.7% lower than the

Table III. Summary of genotype analysisa

Asp299Gly Volunteers Thr399Ile Volunteers

Total Subjects 73 Total Subjects 73 AA (Asp/Asp) 66 CC (Thr/Thr) 69 AG (Asp/Gly) 7 CT (Thr/Ile) 4 GG (Gly/Gly) 0 TT (Ile/Ile) 0

Carrier frequency (95% CI): 0.096 (0.039, 0.1876) Carrier frequency (95% CI): 0.055 (0.015, 0.134) Allele (G) frequency (95% CI): 0.048 (0.0195, 0.0963) Allele (I) frequency (95% CI): 0.027 (0.008, 0.069)

a Three individuals were doubly heterozygous for Asp299Gly and Thr399Ile polymorphisms, one heterozygous for Thr399Ile only, and four heterozygous for Asp299Gly only. In total, there were eight individuals who were heterozygous for one, the other, or both of the two polymorphisms; CI, confidence interval. The Journal of Immunology 1145

adaptive immune responses (39, 40). Therefore, when LPS or its derivatives are used as adjuvants, activation of TLR4 may, in turn, enable the acquired immune response to the immunogen through induction of cytokines and up-regulation of key costimulatory molecules on APCs. Bacterial LPS is a very complex molecule, and its composition varies widely among different species of Gram-negative bacteria. Structural variations, such as the extent of acylation, the length of the acyl chains, and degree of phosphor- FIGURE 6. Cytokine production by PBMC derived from nonpolymor- ylation within the lipid A moiety, and others have been shown to phic volunteers stimulated with synthetic hexaacylated vs pentaacylated affect signaling (5, 6, 8), and likely alter the manner in which these lipid A. Cells from two wild-type individuals were stimulated with 0.5, 1, molecules interact with the TLR4 receptor complex to initiate sig- or 5 ng/ml synthetic hexaacylated (f) or pentaacylated (Ⅺ) lipid A and supernatants collected for cytokine determinations as described in Mate- naling (4). rials and Methods. Results are shown as mean picograms per milliliter Ϯ Consistent with our findings, Hajjar et al. (8) reported that a SD for both volunteers. clinical isolate of P. aeruginosa from a cystic fibrosis patient, that possesses a predominantly hexaacylated lipid A, was a much more potent stimulus in human THP-1 cells and in HEK293 cells trans- levels induced by E. coli LPS in nonpolymorphic and polymorphic fected with human TLR4/MD-2 than a laboratory strain that pos- individuals, respectively (Fig. 5, A and B; percentages not shown sesses a pentaacylated lipid A; yet, both induced equivalent activ- for clarity). Similar findings were observed for IL-1␤, IL-6, and ity in murine cells. However, in contrast to our findings, Hajjar IL-10 (Fig. 5, C–H). Although the suppression was readily ob- et al. (8) found that the differential response they observed was Downloaded from served in both WT and polymorphic individuals, highly significant independent of the species of MD-2 and was attributable to an differences ( p Ͻ 0.01) were recorded only in nonpolymorphic in- 82-aa region in the ectodomain of TLR4 that differs between hu- dividuals (n ϭ 22). This is likely the result of the relatively low man and mouse. This suggests the possibility that some lipid A number of polymorphic individuals evaluated (n ϭ 8). species may be preferentially recognized by human TLR4 (i.e., P. In addition to the previous findings, polymorphic individuals aeruginosa hexaacylated LPS) while other hexaacylated species showed decreased cytokine production in response to both LPS may depend more on their interaction with MD-2 (e.g., E. coli). http://www.jimmunol.org/ preparations when compared with those measured in nonpolymor- Other structural variations may contribute to these apparently con- phic individuals for the same LPS preparation. As shown in Fig. 5, flicting observations: the E. coli lipid A, although hexaacylated, PBMC from polymorphic volunteers secreted lower mean cyto- has a different distribution of amide- and ester-linked fatty acids, kine levels than nonpolymorphic subjects, with up to 90% sup- as well as different length acyl groups, than the hexaacylated P. pression of responsiveness depending on the cytokine and concen- aeruginosa lipid A used in the study by Hajjar et al. (8). Interest- tration evaluated. These differences were observed for all ingly, Kim et al. (41) recently reported the crystal structure of cytokines evaluated, including the immunosuppressive cytokine, murine TLR4-MD-2 complexed with (E5564), a synthetic IL-10. Of note, the decrease in TNF-␣ secreted by PBMC from tetraacylated antagonist of LPS and active lipid A (42). MD-2 was by guest on October 5, 2021 polymorphic individuals was not as pronounced as observed for found to bind to the concave surface of the N-terminal and central the other cytokines (Fig. 5B). This observation may also be attrib- domains of murine TLR4 and the four acyl chains of eritoran oc- utable to the relatively low number of polymorphic individuals cupied nearly 90% of the solvent-accessible volume of a hydro- examined and/or the relatively low level of TNF-␣ that is induced phobic internal pocket in murine MD-2. How hexaacylated and when compared with other cytokines. Overall, these results are pentaacylated LPS and lipid A species interact with this hydro- consistent with and support those obtained in HEK293T cells phobic pocket in situ, and whether a similar pocket is found in transfected with WT or polymorphic TLR4 variants. human MD-2, remain to be determined. In addition, Kim et al. (41) In general, the mean response of PBMC from all volunteers was used gel filtration chromatography to show that in contrast to tet- lower in cells stimulated with S. flexneri 2a LPS, and this was most raacylated eritoran, LPS led to formation of a TLR4/MD-2 het- pronounced at 0.5 and 5 ng/ml LPS (a ϳ20–80% decrease in erotetramer. It is tempting to speculate that hexaacylated vs pen- cytokine production). These findings were confirmed and extended taacylated lipid A species differ in their efficiencies of aggregation in two nonpolymorphic individuals whose PBMC were stimulated of the TLR4/MD-2 complex, with pentaacylated lipid A behaving with synthetic hexaacylated vs pentaacylated lipid A preparations. more similarly to Eritorin (tetraacylated LPS antagonist). As shown in Fig. 6, while high levels of IL-10, IL-1␤, and IL-12 Huber et al. (43) recently reported that rough forms of LPS p70 production were observed in response to hexaacylated lipid A, derived from minnesota or E. coli were much more the response to pentaacylated lipid A remained essentially at back- potent than smooth forms of LPS, suggesting that in addition to ground levels. Similar results were observed for TNF-␣ and IL-6 other lipid A modifications, the core and/or repeating O-Ag production (data not shown). Taken together, the above observa- may constrain the TLR4 response to smooth LPS. In addition, they tions using cells from nonpolymorphic and polymorphic volun- reported that neither lipid A nor rough LPS require CD14 or LPS- teers provide strong evidence to support the contention that both binding protein for signaling, in contrast to smooth LPS. These the acylation state of the LPS and the expression of variant TLR4 findings differ from those of Henricson et al. (31) who showed that molecules may combine to alter significantly cytokine production that lipid A or deep rough mutant LPS preparations are far less following exposure to LPS. stimulatory than the smooth LPS from which they were derived (31). The data presented herein support the findings of Henricson Discussion et al. (31) in that the synthetic lipid As were far less stimulatory The initial interaction of a host with vaccines and/or their adju- than smooth LPS species. The reason for these conflicting obser- vants may significantly impact the overall immunogenicity and vations is not obvious, but may be related to variations in the efficacy of a vaccine. The innate immune response induced by solubility of the LPS preparations and/or the experimental condi- Gram-negative bacterial LPS, a TLR4 agonist, or its derivatives, tions used in these studies (e.g., the absence or presence of has been shown to be responsible and necessary for initiation of in the cultures). 1146 LPS ACYLATION STATE AND TLR4 SNPs ALTER LPS RESPONSIVENESS

In the experiments presented herein, the lipid A was much less vivo. In addition, the relative contribution of TNF-␣ and other potent than intact LPS, and this was most obvious in the HEK293T inflammatory cytokines to the pathogenesis of S. flexneri in the transfectants where neither the synthetic hexaacylated nor the pen- human gut mucosal microenvironment is not well-understood; taacylated lipid A preparations activated TLR4 transfectants, even however, the fact that marked differences have been reported in the at concentrations as high as 8 ␮g/ml (Fig. 3A). It has been shown degree of lipid A acylation among S. flexneri serotypes may help, that MD-2 is an essential coreceptor in LPS-induced TLR4 sig- in part, explain the differences reported in the literature in patho- naling (32). Initially, LPS-binding protein facilitates the transfer of genicity studies in which different Shigella serotypes have LPS/lipid A to CD14. In turn, the lipid A is transferred to MD-2 been used. and this complex interacts with TLR4 to initiate activation (44, Arbour et al. (10) demonstrated that inheritance of two SNPs 45). Increasing the expression of human MD-2 compensated for that encode amino acid substitutions in the ectodomain of TLR4 the failure of hexaacylated lipid A, but not the pentaacylated lipid (Asp299Gly and Thr399Ile) were associated with LPS hyporespon- A, to signal, while murine MD-2 did not differentiate between the siveness. Recently, we demonstrated that expression of TLR4 con- two lipid A species and facilitated signaling by both (Fig. 3, B and structs that carry the Asp299Gly and/or Thr399Ile extracellular C). Our results support earlier predictions that a conically shaped polymorphisms in HEK293T cells result in diminished host re- hexaacylated lipid A is biologically active, whereas a cylindrically sponses to three structurally unrelated TLR4-specific agonists: shaped pentaacylated/tetraacylated lipid A is not active in human Gram-negative LPS, the fusion (F) protein of respiratory syncytial cells (6) and, perhaps, this reflects the differences in the abilities of virus, and Chlamydia heat shock protein 60 (3). In this study, we these two lipid A species to interact with human MD-2. have used both transiently transfected HEK293T cells and PBMC In contrast to our findings using human TLR4-expressing to show that these common, naturally occurring TLR4 polymor- HEK293T transfectants or human PMBC cultures (Fig. 6), mouse phisms that affect ϳ6–10% of the population influence the induc- Downloaded from peritoneal macrophages, when treated with hexaacylated or pen- tion of cytokines in response to both E. coli LPS and S. flexneri 2a taacylated lipid A, secreted similar levels of proinflammatory cy- LPS preparations. Both LPS preparations induced a significantly tokines (Table I), in a dose-dependent manner for IL-1␤ secretion lower response in the doubly polymorphic TLR4 variant (Fig. 4). (Table II). These results clearly support the hypothesis that the Similar results were obtained with PBMC, where polymorphic in- acylation state of lipid A may contribute to species specificity. This dividuals showed decreased cytokine production (e.g., TNF-␣, IL- was originally observed with tetraacylated LPS or lipid A species 1␤, IL-6, and IL-10) in response to both LPS preparations when http://www.jimmunol.org/ in which murine macrophages were responsive, while human cells not compared with those derived from nonpolymorphic individuals for only failed to respond, but the response to active LPS was antag- the same LPS preparation (Fig. 5). Therefore, because polymor- onized by the tetraacylated species (33). More recent studies phic individuals cannot mount an optimal innate immune response demonstrated that murine MD-2 rescues the response to tetraacy- to LPS, it is likely that their ability to respond to vaccines in which lated lipid IVA in a human system (8, 34). Previous studies (35, 46) the LPS is intrinsic to the vaccine itself (e.g., a Shigella vaccine), have shown that activation of TLR4 by taxol is also species-spe- or a vaccine to which an LPS-based adjuvant has been added, may cific, and can be restored by mouse, but not human, MD-2. Our be compromised. findings support the hypothesis that murine MD-2 has a greater Collectively, our data provide direct evidence for the prediction by guest on October 5, 2021 propensity for interaction with synthetic pentaacylated lipid A than that in humans, S. flexneri 2a LPS would elicit a weaker TLR4- human MD-2 and, perhaps, accounts for the observed differences mediated response than E. coli LPS due to differences in the ac- in human vs murine responses. It is tempting to speculate that the ylation status of their lipid A moieties (Fig. 1, B vs A), and that striking differences in the inflammatory responses observed be- both LPS species elicit a diminished response in cells that express tween mouse and human cells might contribute, at least in part, to polymorphic TLR4 variants. In both TLR4 transfectants and in the marked host restriction of Shigella infections to humans and monocytic cultures derived from healthy volunteers, we observed nonhuman primates. The intensity and characteristics of the in- similar trends: pentaacylated/tetraacylated LPS was consistently flammatory responses observed in rodent macrophages might al- less stimulatory than hexaacylated LPS (Fig. 2) and this was more low them to control infection at an early stage, while the relatively striking when synthetic hexaacylated or pentaacylated lipid A mol- meager inflammatory responses of human cells, secondary to the ecules were used as stimuli. Moreover, in transfectants or PBMC failure of the human TLR4 signaling complex to respond robustly expressing polymorphic TLR4 variants, the response to either LPS to hypoacylated S. flexneri 2a LPS lipid A, may be insufficient to or lipid A was further diminished. In sum, we have provided direct control the development of disease. evidence that at least two independent parameters, i.e., the acyla- D’Hauteville et al. (16) previously reported that WT S. flexneri tion state of the LPS itself and the responsiveness of the vaccinee serotype 5a possessed a predominantly hexaacylated lipid A to the LPS contained within the vaccine, can markedly affect im- (93%), in contrast to our S. flexneri 2a (Fig. 1B). Interestingly, mune responses to LPS and, likely, other vaccine Ags. These are deletion of S. flexneri 5a msbB1 or msbB2 genes resulted in de- important considerations that could lead to the development of creased levels of hexaacylated lipid A (to 59 and 73%, respec- more effective vaccines and adjuvants. tively) and a concomitant increase in pentaacylated lipid A (to 34 and 19%, respectively). In the doubly mutated strain, there was no Acknowledgments detectable hexaacylated lipid A with pentaacylated (86%) and tet- We thank Ashley S. Beasley and Robert J. Cotter, Johns Hopkins Univer- raacylated (14%) lipid A species. The extent of histologic damage sity School of Medicine, for carrying out MS analysis of lipid A induced by infection with S. flexneri 5a WT and mutant strains in preparations. a rabbit intestinal loop model roughly paralleled the proportion of hexaacylated lipid A recovered from each strain. Although these Disclosures data have been interpreted to suggest that the level of hexaacylated The authors have no financial conflict of interest. lipid A may influence the extent to which the intestine will be damaged during infection, it is also possible that a preferential References enrichment of inactive or antagonistic tetraacylated lipid A in the 1. Rietschel, E. T., H. Brade, O. Holst, L. Brade, S. Muller-Loennies, U. Mamat, doubly mutated strain might also contribute to its attenuation in U. Zahringer, F. Beckmann, U. Seydel, K. Brandenburg, et al. 1996. Bacterial The Journal of Immunology 1147

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