Differential Induction of the Toll-Like Receptor 4-MyD88-Dependent and-Independent Signaling...

INFECTION AND IMMUNITY, May 2005, p. 2940–2950 Vol. 73, No. 50019-9567/05/$08.00�0 doi:10.1128/IAI.73.5.2940–2950.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Differential Induction of the Toll-Like Receptor 4–MyD88-Dependentand -Independent Signaling Pathways by Endotoxins

Susu M. Zughaier,1* Shanta M. Zimmer,1 Anup Datta,4 Russell W. Carlson,4and David S. Stephens1,2,3

Division of Infectious Diseases, Department of Medicine,1 and Department of Microbiology and Immunology,2 Emory UniversitySchool of Medicine, and Laboratories of Microbial Pathogenesis, Department of Veterans Affairs Medical Center,3

Atlanta, and Complex Carbohydrate Research Center, University of Georgia, Athens,4 Georgia

Received 25 October 2004/Returned for modification 22 November 2004/Accepted 17 January 2005

The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptorcomplex is directly related to lipid A structure or configuration. Endotoxin structure may also influenceactivation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility,human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells werestimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previ-ously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e.,indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-�), interleukin-1� (IL-1�), monocyte che-moattractant protein 1 (MCP-1), macrophage inflammatory protein 3� (MIP-3�), and the MyD88-indepen-dent molecules beta interferon (IFN-�), nitric oxide, and IFN-�-inducible protein 10 (IP-10). Escherichia coli55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations inducedcomparable levels of TNF-�, IL-1�, and MIP-3�, but significantly less IFN-�, nitric oxide, and IP-10. Incontrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-�,nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-� and MIP-3� in time course anddose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected humanembryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significantreduction of TNF-� release but did not influence nitric oxide release. IFN-� polyclonal antibody and IFN-�/�receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist ofboth the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of humanmacrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectivelyinduced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

Bacterial endotoxins of human pathogens interact with mac-rophages and other cells via the Toll-like receptor 4 (TLR4)-MD-2 receptor complex (TLR4 complex) (13), resulting incellular activation, the release of cytokines, chemokines, reac-tive oxygen species, nitric oxide (4) and, in some individuals,the clinical picture of sepsis. However, other endotoxins oftenfound in commensal species act as antagonists of the TLR4complex (14, 44).

Two signaling pathways have been described followingTLR4 activation, the MyD88-dependent (9, 26) and –indepen-dent pathways (15, 33). These signaling pathways depend onToll/interleukin-1 receptor adaptor proteins including MyD88,Mal/TIRAP (10, 17), TRIF/TICAM-1 (32, 47), and TRAM/TICAM-2 (11, 48). Endotoxin activation of the MyD88-depen-dent pathway results in rapid NF-�B activation and release ofproinflammatory cytokines such as tumor necrosis factor alpha(TNF-�), interleukin (IL-)1�, IL-6, and chemokines like mono-cyte chemoattractant protein 1 (MCP-1), macrophage inflamma-tory protein 3� (MIP-3�), and IL-8. Endotoxin activation of the

MyD88-independent pathway results in rapid activation of inter-feron regulatory factor 3 (IRF3) (22, 33) leading to beta inter-feron (IFN-�) release but delayed NF-�B activation (15). IFN-�binds to the type 1 IFN-�/� receptor, which activates STAT1 (1,23, 36) and leads to type 1 IFN-�/�, IFN-�-inducible protein 10(IP-10), MCP-5, RANTES, and nitric oxide release (22, 35, 46).Further, TLR4 activation by endotoxin via the MyD88-indepen-dent (TRIF/TICAM-1) pathway is important for dendritic cellmaturation and provides an important link between the innateand adaptive immune responses (16).

How endotoxin interacts with the TLR4 complex moleculesand recruits specific adaptor proteins remains poorly under-stood. Lipid A structure and configuration of endotoxin areknown to determine the agonist or antagonist activity (24, 27,37). For example, the KDO2-lipid A structure of meningococ-cal lipooligosaccharide (LOS) is necessary for the maximalagonist activation of macrophages via the TLR4 complex (51).The aim of the study was to further determine the influence ofendotoxin structure on activation of the TLR4-MyD88-depen-dent and -independent signaling pathways.

MATERIALS AND METHODS

Reagents. RPMI 1640 medium, Dulbecco’s modified Eagle’s medium, fetalbovine serum, penicillin/streptomycin, sodium pyruvate, and nonessential amino

* Corresponding author. Mailing address: Division of InfectiousDiseases, Emory University School of Medicine, VAMC (I-151), 1670Clairmont Rd, Atlanta, GA 30033. Phone: (404) 321 6111, ext 7454.Fax: (404) 329 2210. E-mail: [email protected].

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acids were obtained from Cellgro Mediatech (Herndon, VA). Phorbol myristateacetate was purchased from GibcoBRL (Grand Island, NY). Human and mouseTNF-�, IFN-� enzyme-linked immunosorbent assay (ELISA) kits were fromR&D systems (Minneapolis, MN). The RAW 264 and 7, 23ScCr (TLR4-defi-cient) cell lines were purchased from the American Type Culture Collection. TheTHP-1 cell line was provided by Fred Quinn (Centers for Disease Control andPrevention, Atlanta, GA). The U937 cell line was obtained from Yusof AbuKwaik (University of Kentucky School of Medicine, Lexington, KY). The MM6cell line was provided by Geert-Jan Boon (Complex Carbohydrate ResearchCenter, University of Georgia, Athens, GA). The C3H/HeJ (TLR4�/�) cell linewas a gift from Bruce Beutler (Scripps Research Institute, La Jolla, CA). Anti-interferon-�/� receptor 1 was purchased from Sigma (Saint Louis, Missouri).Rabbit anti-mouse IFN-� and anti-mouse IFN-� polyclonal antibodies werepurchased from PBL Biomedical Laboratories via R&D Systems.

LOS and LPS purification and quantitation. Endotoxin (lipooligosaccharide,LOS) from the serogroup B Neisseria meningitidis strain NMB (L2 immunotype)and strains 981 (L5 immunotype) and 44/76 (L3 immunotype) was extracted bythe phenol-water method (34). Enteric lipopolysaccharide (LPS) (Sigma Chem-icals, St. Louis, MO) used were Escherichia coli 055:B5, E. coli 111:B4, Serratiamarcescens, Vibrio cholerae Inaba 569B (phenol-water extraction), Pseudomonasaeruginosa 10, and Klebsiella pneumoniae (trichloroacetic acid extraction), Sal-monella enterica serovar Typhimurium TV119 Ra mutant, and Salmonella en-terica serovar Minnesota Re595 mutant (phenol-chloroform-petroleum etherextraction). These endotoxin preparations were further purified and quantified(19, 51). Briefly, residual membrane phospholipids (unsaturated fatty acyl resi-dues C18:0) were removed by repeated extraction of the dry LPS or LOS sampleswith 9:1 ethanol:water. The expected fatty acyl components of 3-OHC12:0,3-OHC14:0 and C12:0 and the absence of membrane phospholipids was assessedby mass spectroscopy.

Purified LPS and LOS samples were standardized based on the number oflipid A molecules per sample (50). The structure of the lipid A of these endo-toxins is noted in Table 1. There are two molecules of �-hydroxymyristic acid permolecule of meningococcal lipid A and four molecules per E. coli lipid A andother LPSs (51). Pseudomonas aeruginosa lipid A was quantified based on thepresence of 3-OHC12:0 since it lacks �-hydroxymyristic acid. All endotoxins(LPS) were resuspended in pyrogen-free water with 0.5% triethylamine, vortexedfor at least 5 min, boiled for 1 h at 65°C, then sonicated for 30 min in a water bathsonicator (L&R, Transisitor/Ultrasonic T-14) to enhance solubility. Endotoxinstock solutions were made up in pyrogen-free water at 10 nmol lipid A/mlconcentration and further diluted with phosphate-buffered saline to 1 nmol lipidA/ml and 100 pmol lipid A/ml with extensive vortexing and sonication prior toeach dilution. Endotoxin samples were used at concentrations ranging from 10 to0.0046 pmol lipid A/ml.

Lipid A purification. To extract lipid A, LOS and LPS were hydrolyzed withharsh acid conditions. Briefly, 50 �l of LOS or LPS (stock concentration 10 nmollipid A/ml) was mixed with 450 �l of 1% acetic acid (pH 2.8) or PBS (pH 7.4),all pyrogen-free solutions, to give final endotoxin concentration of 1 nmol lipidA/ml. After vigorous mixing all tubes were incubated at 90°C for 45 min thendried in a SpeedVac (Savant, Farmingdale, NY). The dried pellets containinglipid A were resuspended in 500 �l of pyrogen free water, vortexed vigorously,sonicated and saved for further use. Lipid A structures were confirmed after acidhydrolysis using thin layer chromatography (12) with chloroform-methanol-wa-ter-triethylamine, 30:12:2:0.1, vol/vol.

Cell cultures. U937, MM6, and THP-1 human macrophage-like cell lines weregrown in RPMI 1640 with L-glutamate supplemented with 10% fetal bovine

serum, 50 IU/ml of penicillin, 50 �g/ml of streptomycin, 1% sodium pyruvate,and 1% nonessential amino acids. Culture flasks were incubated at 37°C withhumidity under 5% CO2. Murine macrophages (RAW 264.7, 23ScCr, and C3H/HeJ) and HEK293 human kidney epithelial cells were grown in Dulbecco’smodified Eagle’s medium supplemented and incubated as mentioned above.

Cytokine induction by LOS and LPS. U937, MM6, and THP-1 human mono-cytes were differentiated into macrophage–like cells using phorbol myristateacetate at a final concentration of 10 ng/106 cell and incubated at 37°C for at least24 h. Differentiated U937 cells express TLR4 but not TLR2 on the surface, whileTHP-1 cells express both receptors (49). Freshly differentiated macrophageswere washed with PBS, counted, and adjusted to 106 cell/ml, transferred into a24-well tissue culture plate (1 ml/well), stimulated with LOS or LPS in dose-response or time course manner and incubated at 37°C with 5% CO2. Cellculture supernatants were harvested and saved at �20°C. Harvested superna-tants from previously stimulated macrophages (containing released cytokinesand chemokines) were subsequently used to stimulate human and murine mac-rophages in a dose-dependent manner (indirect cellular activation).

Cytokine and chemokine quantification by ELISA. Human TNF-�, IL-1�,MCP-1, MIP-3�, and IP-10 Duoset kits (R&D Systems) were used for cytokinequantification according to the manufacturer’s instructions. Maxisorp ELISAplates were obtained from Nalge Nunc International, Rochester, NY.

Nitric oxide induction by murine macrophages. Adherent RAW 246.7 or23ScCr (TLR4-deficient) macrophages were washed with PBS and incubatedwith 5 ml of trypsin for 5 min at 37°C. Harvested cells were washed and resus-pended in Dulbecco’s complete medium. One million macrophages/ml weretransferred to a 24-well tissue culture plate, stimulated with 0.56 pmol lipid A/mlof LOS and incubated overnight. RAW 264.7 and 23ScCr macrophages were alsoindirectly stimulated overnight with serial fold dilutions of 50 �l of cell culturesupernatants from previously stimulated U937, MM6, or THP-1 human macro-phage-like cells exposed to 0.56 pmol lipid A/ml endotoxin. The indirect nitricoxide assay was used to reflect the activity of LPS/LOS in the initial macrophageassay to activate via the MyD88-independent pathway. TLR4-deficient cells wereused to eliminate the contribution of residual LPS in supernatants used in theindirect nitric oxide assay. The induced RAW 264.7 or 23ScCr macrophages wereincubated overnight at 37°C with 5% CO2 and supernatants were harvested andsaved. Nitric oxide release was quantified using the Greiss chemical method aspreviously described (51).

Blocking MyD88-dependent pathway. THP-1 monocytes seeded in 12-wellplates (5 � 105 cells/well) were transiently transfected with 0.5 �g/well of thepDNMyD88 plasmid (Generous gift from Andrew Gewirtz, Emory University)and SuperFect transfection reagent (QIAGEN, Inc., Valencia, CA) for 3 h.Fresh medium was applied and the cells were incubated for 12 to 18 h. The cellswere then stimulated with 0.56 pmol lipid A/ml of endotoxin for 24 h, andsupernatants were harvested and stored. TNF-� production was measured byELISA and indirect (50 �l supernatants were used to stimulate RAW 264.7macrophages for nitric oxide release) nitric oxide was measured by the Greissmethod.

HEK293 transfection with TLR4 and MD-2. HEK293 cells seeded in 12-wellplates (5 � 105 cells/well) were transiently transfected with 0.5 �g/well of thepEFBOS-human TLR4, the pEFBOS-human MD-2, or both plasmids together(Generous gift from Kensuke Miyake, Osaka University) and SuperFect trans-fection reagent (QIAGEN, Inc., Valencia, CA) for 3 h. An empty vector wasused as a control. Fresh media was applied and the cells were recovered for 12to 18 h. The cells were then stimulated with 0.56 pmol lipid A/ml of bacterialendotoxin for 24 h, and IL-8 production was measured by ELISA.

TABLE 1. Lipid A structure of the endotoxins used in this studya

LPS Lipid A phosphorylation Fatty acyl chainsymmetry

Fatty acyl chainnumber 3-OHC10:0 C12:0 3-OHC12:0 C14:0 3-OHC14:0 C16:0

N. meningitidis Bisphosphorylated Symmetrical Hexa-acylated 2 2 2V. cholerae Bisphosphorylated Symmetrical Hexa-acylated 2 2 2E. coli 55:B5 Bisphosphorylated Asymmetrical Hexa-acylated 1 1 4S. enterica serovar Minnesota Bisphosphorylated Asymmetrical Hepta-acylated 1 1 4 1S. enterica serovar Typhimurium Bisphosphorylated Asymmetrical Hepta-acylated 1 1 4 1K. pneumoniae Bisphosphorylated Asymmetrical Hepta-acylated 2 4 1P. aeruginosa Bisphosphorylated Asymmetrical Penta-acylated 1 3 1S. marcescens Bisphosphorylated Asymmetrical Hexa-acylated 1 1 4

a N. meningitidis and V. cholerae lipid A are symmetrical hexa-acylated (29, 30), E. coli lipid A is asymmetrical hexa-acylated (31, 32), Klebsiella pneumoniae,Salmonella enterica serovars Typhimurium and Minnesota lipid A’s are asymmetrical and hepta-acylated (33–35). Pseudomonas aeruginosa lipid A is an asymmetricalpenta-acylated structure (36) and Serratia marcescens lipid A is an asymmetrical hexa-acylated structure (37).

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IFN-�/� receptor blocking. Freshly grown THP-1 cells were washed and ad-justed to 106 cell/ml, resuspended in 200 �l of PBS final volume. IFN-�/�interferon receptors were blocked with 1, 2, 5, or 10 �g/106 cells of the anti-human IFN- �/� receptor 1 polyclonal antibody [anti-human IFN-�/� receptor 1antibody was produced in goats immunized with purified recombinant humanIFN-�/� receptor 1 extracellular domain and the specific IgG was purified byhuman IFN-�/� receptor 1 affinity chromatography (Sigma)]. The cell mixturewas incubated for 30 min at 37°C and then the volume was adjusted to 1 ml withRPMI 1640 supplemented as noted above. Blocked and unblocked cells werestimulated with 1 pmol lipid A/ml of LPS or 10 pmol/ml of lipid A (acidhydrolyzed from meningococcal LOS) and incubated overnight; 100 �l from theharvested supernatants was used to measure the amount of released IP-10induced directly or nitric oxide induced indirectly (50 �l supernatants were usedto stimulate RAW 264.7 macrophages for nitric oxide release). An isotypecontrol antibody (for the human IFN-�/� receptor 1 antibody) normal goat IgGobtained from naı̈ve goat and purified by protein G affinity chromatography waspurchased from R&D Systems, catalog number AB-108-C.

Exogenous IFN-� and IFN-�/� neutralization with polyclonal antibodies. Toconfirm the role of IFN-� in inducing nitric oxide in our experimental model,exogenous IFN-� (R&D Systems) was added in decreasing concentrations, 3, 1.5,0.75, 0.38, and 0.19 ng/106 RAW 264.7 macrophages. The cells were incubatedfor 24 h at 37°C and nitric oxide release was measured in supernatants by theGreiss method. To further investigate the role of IFN-�/� in nitric oxide induc-tion, anti-mouse IFN-� and anti-mouse IFN-� neutralizing polyclonal antibodies(obtained from R&D Systems) were used. RAW 264.7 cells were stimulatedeither directly with LPS (1 pmol lipid A/ml) or lipid A (10 pmol lipid A/ml) orindirectly with 50 �l supernatant (from previously stimulated cells containingreleased cytokines) in the presence or absence of IFN-�/� neutralizing poly-clonal antibodies (2 �g/well). Induced cells were incubated for 24 h at 37°C.Nitric oxide release was measured in supernatants as mentioned above. Anisotype control antibody (for the mouse IFN-� antibody) normal rabbit poly-clonal IgG obtained from naı̈ve rabbit and purified by protein G affinity chro-matography was purchased from R&D Systems, catalog number AB-105-C.

TLR4 and CD14 inhibition. TLR4 and CD14 receptors were blocked withspecific monoclonal antibodies prior to stimulation with endotoxin. U937 andTHP-1 cells were differentiated and prepared as above. One million cells wereresuspended in 200 �l of PBS with 5 �g or 10 �g of anti-TLR4 (gift from KenskuMiyake, Saga, Japan) or anti-CD14 (R&D Systems) added alone or in combi-nation and incubated for 30 min at 37°C with gentle shaking. An isotype controlantibody (for the TLR4 antibody) was used as a control. Cells were then cen-

trifuged at 2,000 rpm (500 � g) for 5 min and resuspended in 1 ml of RPMI 1640medium, stimulated with 0.56 pmol lipid A/ml of endotoxin, and incubatedovernight.

Statistical analysis. Mean values � standard deviation and P values (Studentt test) of at least four independent determinations were calculated with MicrosoftExcel software.

RESULTS

Differential agonist activity of endotoxins. The agonist ac-tivity of endotoxins for the MyD88-dependent and -indepen-dent signaling pathways was first assessed in THP-1 cells (106

cells/ml) constitutively expressing TLR4/MD-2 (Fig. 1 and 2).THP-1 cells were stimulated with highly purified endotoxinsfrom N. meningitidis, E. coli 55:B5, Serratia marcescens, Kleb-siella pneumoniae, Salmonella enterica serovar Typhimurium,Salmonella enterica serovar Minnesota, Pseudomonas aerugi-nosa, and Vibrio cholerae. Cytokine (TNF-�, IL-1�, IP-10, andIFN-�) chemokine (MCP-1 and MIP-3�), and nitric oxideinduction or activity was determined. TNF-�, IL-1�, andMIP-3� release reflected MyD88-dependent pathway activa-tion while IFN-�, IP-10, and nitric oxide reflected MyD88-independent pathway activation (42).

N. meningitidis endotoxin was consistently the most potentactivator of the MyD88-dependent TLR4 pathway (Fig. 1).Equal molar amounts of V. cholerae and E. coli 55:B5 LPS(0.56 pmol lipid A/ml [1 ng/ml] concentrations of lipid A)also induced high levels of TNF-� and MIP-3�. S. marcescens,K. pneumoniae, S. enterica serovar Typhimurium, S. entericaserovar Minnesota, and P. aeruginosa endotoxins inducedTNF-� and MIP-3� above controls, but those levels were sig-nificantly less than with N. meningitidis, E. coli 55:B5, and V.cholerae endotoxins (P 0.001). IL-1� and MCP-1 profiles(data not shown) gave results similar to those obtained forTNF-�.

FIG. 1. TNF-� and MIP-3� (MyD88-dependent) induction by endotoxin. Neisseria meningitidis LOS and E. coli 55:B5 and Vibrio cholerae LPSsinduced more TNF-� and MIP-3� compared to Serratia marcescens, Salmonella enterica serovar Minnesota, Salmonella enterica serovar Typhi-murium, Klebsiella pneumoniae, and Pseudomonas aeruginosa LPSs. *, P 0.001. TNF-� (solid bars) and MIP-3� (dashed bars) induction in 106

THP-1 cells stimulated with highly purified LOS or LPS (0.56 pmol lipid A/ml) for 18 h is shown. Error bars represent standard deviation fromthe average of four readings. The figure is representative of six independent experiments.

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Compared to other endotoxins, N. meningitidis LOS was themost active inducer of nitric oxide via TLR4 (Fig. 2A). Nitricoxide induction was highest from RAW 264.7 macrophagesstimulated directly with 0.56 pmol lipid A/ml of meningococcalendotoxin or indirectly with supernatants (containing releasedcytokines) harvested from macrophages stimulated with 0.56pmol lipid A/ml of N. meningitidis LOS (Fig. 2A). The Salmo-nella LPSs were also significant inducers of nitric oxide com-pared to the other LPSs (P 0.001) except N. meningitidis. Inaddition, N. meningitidis LOS and Salmonella LPSs inducedsignificantly more IP-10 compared to other endotoxins (Fig.2B). Similar results were seen in 23ScCr macrophages stimu-lated with supernatants from RAW 264.7 macrophages in-duced with LPS. TLR4-deficient cells were used to eliminatethe contribution of residual LPS in supernatants used in theindirect nitric oxide assays. These data suggested the differen-tial induction of the MyD88-dependent and -independent sig-naling pathways by different endotoxins.

The role of TLR4 and MyD88 in the selective induction ofsignaling pathways. Significant direct cytokine, chemokine ornitric oxide responses were not seen with TLR4-deficient celllines (C3H/HeJ, 23ScCr, or HEK293) even at very high dosesof endotoxin, but responses were restored in TLR4-MD-2transfected HEK293 cells (data not shown), indicating that theendotoxins utilized TLR4 for MyD88-dependent and indepen-

dent activation. When the TLR4 and CD14 receptors onTHP-1 cells were blocked with monoclonal antibodies prior tostimulation with N. meningitidis LOS or Salmonella enterica, E.coli 55:B5, and V. cholerae LPSs, significant decreases inTNF-� induction (P 0.001) were seen as expected (data notshown).

To confirm the role of MyD88 in the proinflammatory cy-tokine and chemokine induction, the DNMyD88 construct wastransfected into THP-1 cells and used to block the MyD88-dependent pathway. The presence of DNMyD88 resulted insignificant reduction (P 0.0001) of TNF-� release (Fig. 3A)but did not significantly influence indirect nitric oxide release(Fig. 3B). These data indicate that TNF-� production waslargely MyD88-dependent and nitric oxide production (in-duced selectively by N. meningitidis and S. enterica serovarTyphimurium endotoxins) was the result of MyD88-indepen-dent signaling.

Dose- and time course-dependent differential induction ofthe MyD88-dependent and -independent signaling pathways:The differential induction of the MyD88-dependent and -inde-pendent pathways by endotoxin was further investigated indose and time course assays. Human macrophage-like cell lines(THP-1, U937, and MM6) or murine macrophage RAW 264.7cells were stimulated with a range of concentrations of theendotoxins for 1, 2, 3, 4, 5, 8, and 24 h. Supernatants from

FIG. 2. Nitric oxide and IP-10 (MyD88-independent) induction by endotoxin. N. meningitidis LOS and Salmonella LPS induced significantlymore nitric oxide compared to other endotoxins (P 0.001). A: RAW 264.7 cells (106 cells/ml) stimulated directly (solid bars) with highly purifiedLPS (0.56 pmol lipid A/ml) or indirectly (dashed bars) with 50 �l/well of THP-1 supernatants from cells stimulated with 0.56 pmol lipid A/ml LPSand incubated overnight. Nitric oxide release was determined by measuring nitrite accumulated in supernatants with the Greiss method. Error barsrepresent standard deviation from the average of four readings. Data are representative of eight independent experiments. B: IP-10 induction byendotoxins. THP-1 (106 cells/ml) were stimulated with 0.56 pmol lipid A/ml of highly purified endotoxins and incubated for 18 h. IP-10 release wasquantified in harvested supernatants using an ELISA. Error bars represent standard deviation from the average of four readings. Data arerepresentative of four independent experiments. All P values were *, 0.0001 compared to N. meningitidis LOS or **, 0.005 compared to S.enterica serovar Typhimurium or S. enterica serovar Minnesota LPS.



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previously stimulated cells were used to indirectly stimulateRAW 264.7 or 23ScCr (TLR4-deficient) macrophages. N. men-ingitidis LOS (inducer of high levels of both TNF-�, nitricoxide and IP-10), S. enterica serovar Typhimurium LPS (in-ducer of high levels of nitric oxide and IP-10 but modestTNF-� and other proinflammatory cytokines), and E. coli055:B5 and V. cholerae LPS (inducers of TNF-� and otherproinflammatory cytokines but low levels of nitric oxide andIP-10) were studied in detail in these experiments.

N. meningitidis LOS, E. coli 55:B5, and V. cholerae LPS at 10to 0.0046 pmol lipid A/ml concentrations induced TNF-� re-lease in a dose-dependent manner (Fig. 4A). In contrast, S.enterica serovar Typhimurium LPS was a weak inducer ofTNF-� even at high picomolar concentrations (significant dif-ferences, P 0.006, from 0.156 pmol to 10 pmol lipid A/ml). N.meningitidis LOS and S. enterica serovar Typhimurium LPSinduced high levels of nitric oxide release directly from RAW264.7 cells (Fig. 4B) or indirectly with low concentrations ofsupernatants from previously stimulated THP-1 cells (Fig. 4C).In comparison to N. meningitidis LOS and S. enterica serovarTyphimurium LPS, E. coli 55:B5 and V. cholerae LPS werepoor inducers of direct nitric oxide release at concentrationsbelow 5 pmol (V. cholerae) and 1.25 pmol (E. coli 55:B5). Theamount of E. coli 55:B5 LPS required to directly induce equiv-alent nitrite (50 �M) was 34 to 74-fold more (range seen innine different experiments) and the amount of lipid A of V.

cholerae LPS required was 41 to 136-fold more. High concen-trations of supernatants from E. coli 55:B5 and V. cholerae LPSstimulated cells were required to indirectly induce nitric oxide(Fig. 4C).

In time course experiments, N. meningitidis LOS inducedTNF-� release over 3 h (Figs. 5A). E. coli 55:B5 LPS and V.cholerae LPS also rapidly induced TNF-� within 3 h (Fig. 5A)but the supernatants of these activated cells induced less (P 0.004) nitric oxide at all time points compared to supernatantsfrom cells stimulated with N. meningitidis or S. enterica serovarTyphimurium LPS (Fig. 5B). S. enterica serovar TyphimuriumLPS generated minimal TNF-� at all time points (Fig. 5A), butthe supernatants of these cells induced maximal concentrationsof nitric oxide (Fig. 5B).

For both dose-dependent and time course assays, similarresults were obtained in indirect assays when TLR4-deficientmacrophages 23ScCr cells were induced with supernatantsfrom TLR4�/� cells previously stimulated with LPS to elimi-nate the contribution of residual LPS. Similar results were alsoobtained when U937 or MM6 macrophages were used (datanot shown). These data indicate that at identical picomolarconcentrations of lipid A, N. meningitidis LOS was a potentagonist of both the MyD88-dependent and -independent sig-naling pathways. Salmonella LPS selectively induced theMyD88-independent pathway, while E. coli 55:B5 and V. chol-erae LPS induced the MyD88-dependent pathway. Further, a

FIG. 3. Inhibition of MyD88-dependent signaling pathway with DNMyD88. A: TNF-� induction from THP-1 cells alone (solid bars) or cellstransfected with DNMyD88 (dashed bars) stimulated with 5.6 pmol lipid A/ml of endotoxin and incubated for 18 h (P 0.001). B: Indirect nitricoxide induction in RAW 264.7 cells stimulated with 50 �l of supernatants harvested from cells in panel A. Error bars represents the standarddeviation from the average of 4 readings. This figure is representative of three independent experiments. Similar results were obtained when theexperiments were performed at higher (112 pmol/ml) and lower (0.56 pmol/ml) endotoxin doses.



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FIG. 4. Dose-dependent induction of TNF-� and nitric oxide release by endotoxin. A: TNF-� induction from THP-1 cells stimulated with serialfold dilutions of N. meningitidis LOS and S. enterica serovar Typhimurium, E. coli 55:B5 and V. cholerae LPSs (10–0.0046 pmol/ml) and incubatedfor 18 h. B: Direct nitric oxide induction from RAW 264.7 cells stimulated with serial fold dilutions of endotoxins (10–0.0046 pmol lipid A/ml) andincubated for 18 h. C: Indirect nitric oxide induction in RAW 264.7 cells stimulated with serial fold dilutions of supernatants (50 �l to 0.046 �l/well)harvested from THP-1 cells previously stimulated with 0.56 pmol lipid A/ml LPS for 2 h. Error bars represent the � standard deviation from theaverage of 4 readings. *, P values indicate significant differences compared to N. meningitidis LOS. This figure is representative of six independentexperiments. N. meningitidis LOS, diamonds; S. enterica serovar Typhimurium LPS, squares; E. coli 55:B5 LPS, triangles; V. cholerae LPS, crossedsquares.



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soluble mediator other than endotoxin was present in super-natants from endotoxin-exposed cells and this mediator in-duced indirect nitric oxide release.

The role of IFN-� in the differential induction of theMyD88-independent (TRIF) pathway. Induction of theMyD88-independent pathway is postulated to act throughIFN-� (11). Exogenous IFN-� when added to RAW 264.7 or23ScCr macrophages induced nitric oxide in a dose-dependentmanner (Fig. 6A). When the IFN-�/� receptor was blockedwith IFN-�/� receptor 1 antibody, a significant reduction (P 0.0001) in nitric oxide release was observed (Fig. 6B). In ad-dition, anti-IFN-� polyclonal antibody when added to RAW264.7 cells directly stimulated with N. meningitidis LOS at con-centrations of 1 to 0.031 pmol lipid A/ml significantly reduced(P 0.0005) nitric oxide induction (Fig. 6C). No decrease innitric oxide release was seen when isotype control antibodiesfor human anti-IFN-�/� receptor 1 or mouse anti-IFN-� poly-clonal antibodies were used (data not shown). When RAW264.7 macrophages induced with 1 pmol LOS nitric oxide re-lease was 66.2 � 1.94 �M nitrite, but in the presence of 2, 4, or6 �g/ml of anti-IFN-� polyclonal antibody, a significant de-

crease in nitric oxide release (47.8 � 1.38, 39.8 � 1.91, and 30.1� 2.4 �M nitrite, respectively) was observed. Similar resultswere seen when anti-IFN-� polyclonal antibody was added to23ScCr cells and stimulated with supernatants from previouslyinduced TLR4�/� cells (data not shown). Further, anti-IFN-�and anti-IFN-� polyclonal antibodies neutralized the effects ofinduced IFN-� and significantly reduced nitric oxide release(Fig. 6D). These data support a role for IFN-� release in thedifferential induction of the MyD88-independent pathway andnitric oxide release. The differential activation of the MyD88-independent pathway by N. meningitidis LOS and SalmonellaLPS was likely due to enhancement of IFN-�/� inductiondownstream of TLR4 signaling and likely reflects an autocrine/paracrine amplification via the IFN-�/� receptor and STAT1pathway activation (31).

DISCUSSION

Ligand (e.g., endotoxin)-triggered activation of TLRs in-volves cytoplasmic TIR domain-containing adaptor proteinsthat are essential for signal transduction. These TIR domain-containing adaptors (MyD88, Mal/TIRAP, TRIF, TRAM, and

FIG. 5. Time course induction of the MyD88-dependent and -independent signaling pathways by endotoxin. A: Induction of TNF-� fromTHP-1 (106 cells/ml) stimulated with 0.56 pmol lipid A/ml of endotoxin overnight. B: Indirect nitric oxide induction from RAW 264.7 cellsstimulated with 50 �l of the same THP-1 supernatants harvested in panel A. N. meningitidis LOS, diamonds; S. enterica serovar Typhimurium LPS,squares; E. coli 55:B5 LPS, triangles; V. cholerae LPS, crossed squares. Error bars represent the standard deviation from the average of fourreadings. This figure is representative of six independent experiments.



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possibly others) provide specificity for the individual TLR-mediated signaling pathways (2, 41). For example, TLR2, -5,and -9 ligands utilize the MyD88-dependent pathway, whileTLR3 activation is MyD88-independent. TLR2 and TLR4MyD88-dependent activation requires Mal/TIRAP (33), TLR4mediated MyD88-independent signaling requires TRIF andTRAM (11, 48), and TLR3 does not utilize MyD88 or TRAMbut requires TRIF (3, 8).

Specifically, TLR4 activation of the MyD88-dependent path-way recruits MyD88 and Mal/TIRAP to the TLR4-TIR do-main resulting in the activation of TRAF6, early phase activa-tion of NF-�B and mitogen-activated protein kinase and thesubsequent induction of proinflammatory cytokines such asTNF-� (18). TLR4-mediated activation of the MyD88-inde-pendent signaling recruits TRIF and TRAM that activateIRF3 and IRF7. This results in the rapid production of IFN-�and consequently IFN-� and other interferon-inducible genessuch as IP-10 and nitric oxide and the late phase activation ofNF-�B and mitogen-activated protein kinases (11).

In this study, differential induction of the TLR4-MD-2MyD88-dependent and -independent signaling pathways withpathophysiologically important concentrations of endotoxinwas demonstrated. TLR4 was required and N. meningitidisLOS was the most potent agonist of both the TLR4-MyD88-dependent and -independent signaling pathways. At equal mo-lar lipid A concentrations, E. coli 55:B5 and V. cholerae LPS

induced the MyD88-dependent pathway and released proin-flammatory cytokines and chemokines such as TNF-� andMIP3� but induced significantly less IFN-�, IP-10, or nitricoxide compared to equal concentrations of meningococcalLOS. In addition, cytokines released in supernatants via theMyD88-dependent pathway by E. coli 55:B5 and V. choleraeLPSs induced significantly less nitric oxide as shown by theindirect induction experiments. In contrast, Salmonella LPSinduced the MyD88-independent signaling pathway mediatorsIFN-�, IP-10, and nitric oxide but low amounts of the proin-flammatory cytokines such as TNF-�. The differential activa-tion of the MyD88-independent pathway was driven by IFN-�/� rather than the major proinflammatory cytokine TNF-�.

The differences in induction of MyD88-dependent and –in-dependent signaling by endotoxin suggests that the initial in-teraction of endotoxin with the extracellular domain of theTLR4 complex results in changes that lead to the recruitmentof different adaptor proteins. Differences in the affinity of en-dotoxin binding to TLR4 and or MD-2 (40, 43) and resultingdifferent conformational changes may create more or less bind-ing sites for specific adaptor proteins on the TIR domain ofTLR4, modulating the signaling pathway. Alternatively, thenumber of TLR4-MD-2 molecules aggregated by endotoxinsmay be different and lead to differences in adaptor proteinbinding to the cytoplasmic domain of TLR4.

The differential induction of MyD88-dependent and -inde-

FIG. 6. Induction of nitric oxide by endotoxin was IFN-� mediated. A: Nitric oxide induction in RAW 264.7 cells stimulated with two folddilutions (3–0.19 ng/ml) of exogenous IFN-�. B: Nitric oxide induction in RAW 264.7 cells stimulated with 50 �l supernatants harvested fromTHP-1 cells (106) blocked with 1, 5, or 10 �g of anti-IFN-�/� receptor 1 polyclonal antibody and stimulated with 10 pmol/ml of N. meningitidis lipidA. Controls were supernatants (50 �l) from unstimulated THP-1 cells, unstimulated RAW 264.7 cells, and lipid A without anti-IFN-�/� receptor1 antibody. C: Direct nitric oxide induction in RAW 264.7 cells stimulated with decreasing doses of wild-type N. meningitidis LOS in the presenceor absence of murine anti-IFN-� polyclonal antibody (2 �g/well). D: Indirect nitric oxide induction in RAW 264.7 cells induced with 50 �l of THP-1supernatants stimulated with 10 pmol/ml of N. meningitidis lipid A. Anti-IFN-� (2 �g/well) or anti-IFN-� (2 �g/well) or both antibodies togetherwere added directly into RAW 264.7 cells. Error bars represent the standard deviation from the average of four readings. This experiment figureis representative of three independent experiments.



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pendent signaling by endotoxins are likely due to differences inlipid A fatty acyl structure or conformation and structure of thelipid A head groups. The number and length of the fatty acylchains, the phosphorylation of the lipid A, and the linkage toketodeoxyoctulosonic acid (KDO) are known to influence bi-ological activity by changing the supramolecular conformationof lipid A (38–40, 43). A bisphosphorylated, C12 and C14,hexa-acylated lipid A structure linked to KDO is the mostbioactive when the MyD88-dependent cytokine TNF-� is themeasure of bioactivity (5, 6, 43).

In agreement with these data, N. meningitidis LOS, E. coli55:B5, and V. cholerae LPS which are hexa-acylated with C12and C14 chain length induced significantly more TNF-� thanhepta-acylated Salmonella and Klebsiella LPS or the penta-acylated P. aeruginosa LPS. Differences in lipid A structurealso influence the binding affinity to the TLR4-MD-2 receptorcomplex (40, 43) and, as shown here, modulate the MyD88-independent (TRIF) signaling pathway. Interestingly, thehexa-acylated lipid A structure of N. meningitidis LOS and thehepta-acylated lipid A structure of Salmonella LPS both dra-matically activate the MyD88-independent (TRIF) pathway.Preliminary fluorescence resonance energy transfer measure-ments data indicate that N. meningitidis LOS and SalmonellaLPS clustered or recruited significantly more TRIF adaptorproteins in lipid rafts of THP-1 cell membrane compared to E.coli 55:B5 and V. cholerae (personal communication, KathyTriantafilou, Sussex University, United Kingdom).

The differential induction by the endotoxins was not due toimpurities of the endotoxin preparations, as the effects wereonly seen when TLR4 was present or functional. These highlypurified preparations were free of protein, contaminatingphospholipids, DNA or RNA and were carefully standardizedbased on the same number of lipid A molecules. The differenteffects of the same concentration of the Salmonella endotoxinson the MyD88-dependent and -independent signaling path-ways in the assays further support these conclusions. Lipid Aextracted by acid hydrolysis is a weaker agonist (28) comparedto unhydrolyzed LPS in activating the TLR4-MyD88-depen-dent and -independent signaling pathways, and thus LPS wasused in this study to reflect the differential induction of TLR4.Moreover, meningococcal KDO2-lipid A was the minimalstructure required for optimal TLR4 complex activation (49).In support, a deep rough Salmonella enterica serovar Minne-sota Re595 LPS (KDO2-lipid A) used in this study was com-pared to a different S. enterica serovar Minnesota Re595 LPSpreparation (gift from Frank Neumann, Bioaxxess, UnitedKingdom) and to Salmonella enterica serovar Typhimurium RaLPS, which consists of complete core sugars. All SalmonellaLPSs induced similar large amounts of nitric oxide but littleTNF-�. Thus, the length and structure of oligosaccharidechains beyond KDO2 had no significant effect on activatingmacrophages.

Differential induction of the TLR4-MyD88-dependent and–independent signaling pathways is supported by earlier in vivoand in vitro studies (29, 30). Netea et al. found that TNFknockout mice were as susceptible as wild type controls to S.enterica serovar Typhimurium LPS, but they were significantlymore resistant to lethal endotoxemia induced by E. coli 055:B5or K. pneumoniae LPS (29, 30). The studies also demonstrateda delayed cytokine response in TNF knockout mice challenged

with S. enterica serovar Typhimurium LPS but not in micechallenged with E. coli LPS (29). In their mouse model, thelethal effects of S. enterica serovar Typhimurium LPS weremediated through cytokines such as IFN-� and IL-18. Mathiaket al. (25) found lipopolysaccharides from different bacterialsources elicit disparate effects on cytokine responses in wholeblood assays. Further, S. enterica serovar Typhimurium ex-pressing a mutant lipid A (lacking a secondary fatty acyl chain)has been shown to induce less TNF-�, IL-1�, inducible nitricoxygen species than wild-type S. enterica serovar Typhimurium(45). However, both the wild-type and the mutant Salmonellastrains were able to induce murine dendritic cell maturation ina multiplicity of infection-independent manner (21).

Hoebe et al. demonstrated that LPS-TLR4 dendritic cellmaturation and upregulation of costimulatory molecules isTRIF-IFN-� axis dependent (16). Studies in TRIF-deficientmice indicate that TRIF is not directly involved in the LPS-induced activation of the MyD88-dependent signaling, how-ever, TRIF-deficient mice are impaired in LPS-induced in-flammatory cytokine production (47). MyD88-deficient micedemonstrated late activation of NF-�B and mitogen-activatedprotein kinases but no impairment in dendritic cell activationor maturation (9, 20). Interaction of the MyD88-dependentand -independent signaling is required for the overall TLR4-mediated inflammatory cytokine response induced by endo-toxin (41).

IFN-�/� release appears to be a key molecule in the differ-ential induction of the MyD88-independent (TRIF) signalingpathway by N. meningitidis LOS and Salmonella LPS. Poly-clonal antibodies to IFN-� and IFN-� and the blocking of theIFN-�/� receptor with anti IFN-�/� receptor 1 antibody mark-edly decreased direct and indirect nitric oxide induction. Fur-ther, when MyD88-dependent signaling was blocked usingDNMyD88, significant reduction in TNF-� release was seen(7), but no effect on nitric oxide release was observed. Thesedata suggest that IFN-�/� was responsible for the differentialinduction of the MyD88-independent (TRIF) signaling path-way by N. meningitidis and Salmonella endotoxins, and is inagreement with previous reports showing TLR4 mediatedSTAT1 phosphorylation was abolished in IFN-�/� receptor1-deficient mice (8), and nitric oxide response was impaired inSTAT1-deficient mice (8, 31).

In conclusion, Salmonella LPSs were found to enhance theactivation of the TLR4-MyD88-independent pathway, whilewith E. coli 55:B5 and V. cholerae LPSs, the TLR4-MyD88-dependent pathway was dominant. N. meningitidis LOS inequal molar lipid A concentrations was a potent inducer ofboth pathways. The differential induction of the TLR4-MyD88-independent pathway was IFN-�-mediated. The dif-ferent activities of these endotoxins may be influenced by af-finity of binding to LBP, CD14, or MD-2 (upstream TLR4signaling), differences in recruitment of TIR adaptor proteinsand activation of the IFN-�/� transcription factors like IRF-3and IRF-7 or other downstream TLR4 signaling moleculessuch as TNF-receptor-associated factor 6 (TRAF6), IL-1 re-ceptor-associated kinases, transforming growth factor beta-ac-tivated kinase (TAK1), and TAK1-binding protein (TAB1),possibilities that are under current investigation.

The contribution of the recruitment of other receptors orunknown adaptor proteins in the differential induction of the



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TLR4 signaling pathways is also an explanation for the results.Triantafilou and Triantafilou (45) have identified a receptorsignaling complex for LPS including TLR4, the integrinsCD11c and/or CD18, as well as CD55, HSP70 and -90,CXCR4, and growth differentiation factor 5. However, thecontribution of coreceptors is dependent upon TLR4, since noresponses to endotoxin are seen in the absence of TLR4. Dif-ferences in MyD88-dependent and –independent signaling byendotoxins may help explain different clinical features of bac-teremia disease caused by gram-negative organisms and differ-ences in immune responses, and may prove useful in the designof adjuvants for vaccines.

ACKNOWLEDGMENTS

This work was supported by grant R01 AI033517-10 from the Na-tional Institutes of Health to D.S.S.

We have no conflicting financial interests.

REFERENCES

1. Akira, S., and K. Hoshino. 2003. Myeloid differentiation factor 88-dependentand -independent pathways in toll-like receptor signaling. J Infect. Dis.187(Suppl. 2):S356–63.

2. Beutler, B. 2004. Innate immunity: an overview. Mol. Immunol. 40:845–859.3. Beutler, B., K. Hoebe, X. Du, E. Janssen, P. Georgel, and K. Tabeta. 2003.

Lps2 and signal transduction in sepsis: at the intersection of host responsesto bacteria and viruses. Scand. J. Infect. Dis. 35:563–567.

4. Beutler, B., K. Hoebe, X. Du, and R. J. Ulevitch. 2003. How we detectmicrobes and respond to them: the Toll-like receptors and their transducers.J. Leukoc. Biol. 74:479–485.

5. Blunck, R., O. Scheel, M. Muller, K. Brandenburg, U. Seitzer, and U. Seydel.2001. New insights into endotoxin-induced activation of macrophages: in-volvement of a K� channel in transmembrane signaling. J. Immunol. 166:1009–1015.

6. Brandenburg, K., S. Kusumoto, and U. Seydel. 1997. Conformational studiesof synthetic lipid A analogues and partial structures by infrared spectroscopy.Biochim. Biophys. Acta. 1329:183–201.

7. Byrd-Leifer, C. A., E. F. Block, K. Takeda, S. Akira, and A. Ding. 2001. Therole of MyD88 and TLR4 in the LPS-mimetic activity of Taxol. Eur. J. Im-munol. 31:2448–2457.

8. Doyle, S. E., R. O�Connell, S. A. Vaidya, E. K. Chow, K. Yee, and G. Cheng.2003. Toll-like receptor 3 mediates a more potent antiviral response thanToll-like receptor 4. J. Immunol. 170:3565–3571.

9. Feng, C. G., C. A. Scanga, C. M. Collazo-Custodio, A. W. Cheever, S. Hieny,P. Caspar, and A. Sher. 2003. Mice lacking myeloid differentiation factor 88display profound defects in host resistance and immune responses to Myco-bacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- andTLR4-deficient animals. J. Immunol. 171:4758–4764.

10. Fitzgerald, K. A., E. M. Palsson-McDermott, A. G. Bowie, C. A. Jefferies,A. S. Mansell, G. Brady, E. Brint, A. Dunne, P. Gray, M. T. Harte, D.McMurray, D. E. Smith, J. E. Sims, T. A. Bird, and L. A. O’Neill. 2001. Mal(MyD88-adapter-like) is required for Toll-like receptor-4 signal transduc-tion. Nature 413:78–83.

11. Fitzgerald, K. A., D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz,B. Monks, P. M. Pitha, and D. T. Golenbock. 2003. LPS-TLR4 signaling toIRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J. Exp.Med. 198:1043–1055.

12. Fukuoka, S., H. Kamishima, Y. Nagawa, H. Nakanishi, K. Ishikawa, Y.Niwa, E. Tamiya, and I. Karube. 1992. Structural characherization of lipid Acomponent of Erwinia corotovora lipopolysaccharide. Arch. Microbiol. 157:311–318.

13. Gioannini, T. L., A. Teghanemt, D. Zhang, N. P. Coussens, W. Dockstader,S. Ramaswamy, and J. P. Weiss. 2004. Isolation of an endotoxin-MD-2complex that produces Toll-like receptor 4-dependent cell activation at pi-comolar concentrations. Proc. Natl. Acad. Sci. USA 101:4186–4191.

14. Hawkins, L. D., W. J. Christ, and D. P. Rossignol. 2004. Inhibition ofendotoxin response by synthetic TLR4 antagonists. Curr. Top. Med. Chem.4:1147–1171.

15. Hoebe, K., X. Du, P. Georgel, E. Janssen, K. Tabeta, S. O. Kim, J. Goode, P.Lin, N. Mann, S. Mudd, K. Crozat, S. Sovath, J. Han, and B. Beutler. 2003.Identification of Lps2 as a key transducer of MyD88-independent TIR sig-nalling. Nature 424:743–748.

16. Hoebe, K., E. M. Janssen, S. O. Kim, L. Alexopoulou, R. A. Flavell, J. Han,and B. Beutler. 2003. Upregulation of costimulatory molecules induced bylipopolysaccharide and double-stranded RNA occurs by Trif-dependent andTrif-independent pathways. Nat. Immunol. 4:1223–1229.

17. Horng, T., G. M. Barton, and R. Medzhitov. 2001. TIRAP: an adaptermolecule in the Toll signaling pathway. Nat. Immunol. 2:835–841.

18. Huang, Q., J. Yang, Y. Lin, C. Walker, J. Cheng, Z. G. Liu, and B. Su. 2004.Differential regulation of interleukin 1 receptor and Toll-like receptor sig-naling by MEKK3. Nat. Immunol. 5:98–103.

19. Kahler, C. M., R. W. Carlson, M. M. Rahman, L. E. Martin, and D. S.Stephens. 1996. Two glycosyltransferase genes, lgtF and rfaK, constitute thelipooligosaccharide ice (inner core extension) biosynthesis operon of Neis-seria meningitidis. J. Bacteriol. 178:6677–6684.

20. Kaisho, T., O. Takeuchi, T. Kawai, K. Hoshino, and S. Akira. 2001. Endo-toxin-induced maturation of MyD88-deficient dendritic cells. J. Immunol.166:5688–5694.

21. Kalupahana, R., A. R. Emilianus, D. Maskell, and B. Blacklaws. 2003.Salmonella enterica serovar Typhimurium expressing mutant lipid A withdecreased endotoxicity causes maturation of murine dendritic cells. Infect.Immun. 71:6132–6140.

22. Kawai, T., O. Takeuchi, T. Fujita, J. Inoue, P. F. Muhlradt, S. Sato, K.Hoshino, and S. Akira. 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 andthe expression of a subset of lipopolysaccharide-inducible genes. J. Immunol.167:5887–5894.

23. Kovarik, P., D. Stoiber, M. Novy, and T. Decker. 1998. Stat1 combinessignals derived from IFN-gamma and LPS receptors during macrophageactivation. EMBO J. 17:3660–3668. (Erratum, EMBO J. 17:4210.)

24. Kusumoto, S., K. Fukase, Y. Fukase, M. Kataoka, H. Yoshizaki, K. Sato, M.Oikawa, and Y. Suda. 2003. Structural basis for endotoxic and antagonisticactivities: investigation with novel synthetic lipid A analogs. J. EndotoxinRes. 9:361–366.

25. Mathiak, G., K. Kabir, G. Grass, H. Keller, E. Steinringer, T. Minor, C.Rangger, and L. F. Neville. 2003. Lipopolysaccharides from different bacte-rial sources elicit disparate cytokine responses in whole blood assays. Int. J.Mol. Med. 11:41–44.

26. Miyake, K. 2004. Innate recognition of lipopolysaccharide by Toll-like re-ceptor 4-MD-2. Trends Microbiol. 12:186–192.

27. Mueller, M., B. Lindner, S. Kusumoto, K. Fukase, A. B. Schromm, and U.Seydel. 2004. Aggregates are the biologically active units of endotoxin.J. Biol. Chem. 279:26307–26313.

28. Muroi, M., and K. Tanamoto. 2002. The polysaccharide portion plays anindispensable role in Salmonella lipopolysaccharide-induced activation ofNF-kappaB through human toll-like receptor 4. Infect. Immun. 70:6043–6047.

29. Netea, M. G., G. Fantuzzi, B. J. Kullberg, R. J. Stuyt, E. J. Pulido, R. C.McIntyre, Jr., L. A. Joosten, J. W. Van der Meer, and C. A. Dinarello. 2000.Neutralization of IL-18 reduces neutrophil tissue accumulation and protectsmice against lethal Escherichia coli and Salmonella typhimurium endotox-emia. J. Immunol. 164:2644–2649.

30. Netea, M. G., B. J. Kullberg, L. A. Joosten, T. Sprong, I. Verschueren, O. C.Boerman, F. Amiot, W. B. van den Berg, and J. W. Van der Meer. 2001.Lethal Escherichia coli and Salmonella typhimurium endotoxemia is medi-ated through different pathways. Eur. J. Immunol. 31:2529–2538.

31. Ohmori, Y., and T. A. Hamilton. 2001. Requirement for STAT1 in LPS-induced gene expression in macrophages. J. Leukoc. Biol. 69:598–604.

32. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, and T. Seya. 2003.TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-me-diated interferon-beta induction. Nat. Immunol. 4:161–167.

33. Oshiumi, H., M. Sasai, K. Shida, T. Fujita, M. Matsumoto, and T. Seya.2003. TIR-containing adapter molecule (TICAM)-2, a bridging adapter re-cruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta. J. Biol.Chem. 278:49751–49762.

34. Rahman, M. M., D. S. Stephens, C. M. Kahler, J. Glushka, and R. W.Carlson. 1998. The lipooligosaccharide (LOS) of Neisseria meningitidis se-rogroup B strain NMB contains L2, L3, and novel oligosaccharides, and lacksthe lipid-A 4�-phosphate substituent. Carbohydr. Res. 307:311–324.

35. Re, F., and J. L. Strominger. 2001. Toll-like receptor 2 (TLR2) and TLR4differentially activate human dendritic cells. J. Biol. Chem. 277:23427–23432.

36. Rhee, S. H., B. W. Jones, V. Toshchakov, S. N. Vogel, and M. J. Fenton. 2003.Toll-like receptors 2 and 4 activate STAT1 serine phosphorylation by distinctmechanisms in macrophages. J. Biol. Chem. 278:22506–22512.

37. Saitoh, S. I., S. Akashi, T. Yamada, N. Tanimura, M. Kobayashi, K. Konno,F. Matsumoto, K. Fukase, S. Kusumoto, Y. Nagai, Y. Kusumoto, A. Kosugi,and K. Miyake. 2004. Lipid A antagonist, lipid IVa, is distinct from lipid Ain interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-inducedTLR4 oligomerization. Int. Immunol. 16:961–969.

38. Schromm, A. B., K. Brandenburg, H. Loppnow, A. P. Moran, M. H. Koch,E. T. Rietschel, and U. Seydel. 2000. Biological activities of lipopolysaccha-rides are determined by the shape of their lipid A portion. Eur. J. Biochem.267:2008–2013.

39. Seydel, U., A. B. Schromm, R. Blunck, and K. Brandenburg. 2000. Chemicalstructure, molecular conformation, and bioactivity of endotoxins. Chem.Immunol. 74:5–24.

40. Stover, A. G., J. Da Silva Correia, J. T. Evans, C. W. Cluff, M. W. Elliott,E. W. Jeffery, D. A. Johnson, M. J. Lacy, J. R. Baldridge, P. Probst, R. J.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Ulevitch, D. H. Persing, and R. M. Hershberg. 2004. Structure-activity rela-tionship of synthetic toll-like receptor 4 agonists. J. Biol. Chem. 279:4440–4449.

41. Takeda, K., and S. Akira. 2004. TLR signaling pathways. Semin. Immunol.16:3–9.

42. Takeda, K., and S. Akira. 2003. Toll receptors and pathogen resistance. CellMicrobiol. 5:143–153.

43. Tamai, R., Y. Asai, M. Hashimoto, K. Fukase, S. Kusumoto, H. Ishida, M.Kiso, and T. Ogawa. 2003. Cell activation by monosaccharide lipid A ana-logues utilizing Toll-like receptor 4. Immunology 110:66–72.

44. Triantafilou, M., K. Brandenburg, S. Kusumoto, K. Fukase, A. Mackie, U.Seydel, and K. Triantafilou. 2004. Combinational clustering of receptorsfollowing stimulation by bacterial products determines lipopolysaccharideresponses. Biochem. J. 381:527–536.

45. Triantafilou, M., and K. Triantafilou. 2002. Lipopolysaccharide recognition:CD14, TLRs and the LPS-activation cluster. Trends Immunol. 23:301–304.

46. Vogel, S., M. J. Hirschfeld, and P. Y. Perera. 2001. Signal integration inlipopolysaccharide (LPS)-stimulated murine macrophages. J. EndotoxinRes. 7:237–241.

47. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O.Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira. 2003. Role ofadaptor TRIF in the MyD88-independent toll-like receptor signaling path-way. Science 301:640–643.

48. Yamamoto, M., S. Sato, H. Hemmi, S. Uematsu, K. Hoshino, T. Kaisho, O.Takeuchi, K. Takeda, and S. Akira. 2003. TRAM is specifically involved inthe Toll-like receptor 4-mediated MyD88-independent signaling pathway.Nat. Immunol. 4:1144–1150.

49. Yang, S., R. Tamai, S. Akashi, O. Takeuchi, S. Akira, S. Sugawara, and H.Takada. 2001. Synergistic effect of muramyldipeptide with lipopolysaccha-ride or lipoteichoic acid to induce inflammatory cytokines in human mono-cytic cells in culture. Infect. Immun. 69:2045–2053.

50. York, W. S., D., A. G., McNeil, M., Stevenson, T. T., Albersheim, P. 1985.Isolation andcharacterization of plant cell walls and cell wall components.Methods Emzymol. 118:3–40.

51. Zughaier, S. M., Y. L. Tzeng, S. M. Zimmer, A. Datta, R. W. Carlson, andD. S. Stephens. 2004. Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway.Infect. Immun. 72:371–380.

Editor: J. D. Clements



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INFECTION AND IMMUNITY, May 2006, p. 3077 Vol. 74, No. 50019-9567/06/$08.00�0 doi:10.1128/IAI.74.5.3077.2006

ERRATUM

Differential Induction of the Toll-Like Receptor 4–MyD88-Dependentand -Independent Signaling Pathways by Endotoxins

Susu M. Zughaier, Shanta M. Zimmer, Anup Datta, Russell W. Carlson,and David S. Stephens

Division of Infectious Diseases, Department of Medicine, and Department of Microbiology and Immunology, Emory UniversitySchool of Medicine, and Laboratories of Microbial Pathogenesis, Department of Veterans Affairs Medical Center,

Atlanta, and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia

Volume 73, no. 5, p. 2940–2950, 2005. Page 2940, column 2, lines 4 and 5: “(1, 23, 36)” should read “(1a, 23, 36).”Page 2941, Table 1: Footnote a should read “The published lipid A structures of these endotoxins showed that meningococcal

and V. cholerae lipid A is a symmetrical hexa-acylated structure (6a, 19a), that E. coli lipid A is an asymmetrical hexa-acylatedstructure (1b, 7a), that Klebsiella pneumoniae, Salmonella serovar Typhimurium, and Salmonella serovar Minnesota lipid A is anasymmetrical hepta-acylated structure (19, 21a, 39a), that Pseudomonas aeruginosa lipid A is an asymmetrical penta-acylatedstructure (8a), and that Serratia marcescens lipid A is an asymmetrical hexa-acylated structure (1).”

Page 2949: The following references should be added. Because of these additions, original reference 1 becomes reference 1a.

1. Adams, G. A., and P. P. Singh. 1970. The chemical constitution of lipid Afrom Serratia marcescens. Can. J. Biochem. 48:55–62.

1a.Akira, S., and K. Hoshino. 2003. Myeloid differentiation factor 88-dependentand -independent pathways in Toll-like receptor signaling. J. Infect. Dis.187(Suppl. 2):S356–S363.

1b.Backhed, F., S. Normark, E. K. Schweda, S. Oscarson, and A. Richter-Dahlfors. 2003. Structural requirements for TLR4-mediated LPS signalling:a biological role for LPS modifications. Microbes Infect. 5:1057–1063.

6a.Broady, K. W., E. T. Rietschel, and O. Luderitz. 1981. The chemical struc-ture of the lipid A component of lipopolysaccharides from Vibrio cholerae.Eur. J. Biochem. 115:463–468.

7a.Chatterjee, S. N., and K. Chaudhuri. 2003. Lipopolysaccharides of Vibriocholerae. I. Physical and chemical characterization. Biochim. Biophys. Acta1639:65–79.

8a.Ernst, R. K., A. M. Hajjar, J. H. Tsai, S. M. Moskowitz, C. B. Wilson, andS. I. Miller. 2003. Pseudomonas aeruginosa lipid A diversity and its recogni-tion by Toll-like receptor 4. J. Endotoxin Res. 9:395–400.

19a.Kahler, C. M., and D. S. Stephens. 1998. Genetic basis for biosynthesis,structure, and function of meningococcal lipooligosaccharide (endotoxin).Crit. Rev. Microbiol. 24:281–334.

21a.Kanegasaki, S., K. Tanamoto, T. Yasuda, J. Y. Homma, M. Matsuura, M.Nakatsuka, Y. Kumazawa, A. Yamamoto, T. Shiba, S. Kusumoto, et al. 1986.Structure-activity relationship of lipid A: comparison of biological activitiesof natural and synthetic lipid A’s with different fatty acid compositions.J. Biochem. (Tokyo) 99:1203–1210.

39a.Silipo, A., R. Lanzetta, A. Amoresano, M. Parrilli, and A. Molinaro. 2002.Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOFmass spectrometry analysis of lipid A fatty acid distribution. J. Lipid Res.43:2188–2195.

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