of March 27, 2018.This information is current as

Models of Inflammatory Bowel DiseaseAnti-Inflammatory Effects in Two Murine A Synthetic TLR4 Antagonist Has

Fling and Robert M. HershbergBielefeldt-Ohmann, Peter Probst, Eric Jeffery, Steven P. Richard J. Ulevitch, David H. Persing, HelleJean da Silva Correia, David A. Johnson, R. Thomas Crane, Madeline M. Fort, Afsaneh Mozaffarian, Axel G. Stöver,

http://www.jimmunol.org/content/174/10/6416doi: 10.4049/jimmunol.174.10.6416

2005; 174:6416-6423; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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A Synthetic TLR4 Antagonist Has Anti-Inflammatory Effectsin Two Murine Models of Inflammatory Bowel Disease1

Madeline M. Fort,* Afsaneh Mozaffarian,* Axel G. Stover,2* Jean da Silva Correia,†

David A. Johnson,‡ R. Thomas Crane,‡ Richard J. Ulevitch,† David H. Persing,*§

Helle Bielefeldt-Ohmann,¶ Peter Probst,* Eric Jeffery,* Steven P. Fling,3* andRobert M. Hershberg4*§

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel diseaseis due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonisticactivity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proin-flammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularlysecondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-� release. Importantly,treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: thedextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between entericbacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease. TheJournal of Immunology, 2005, 174: 6416–6423.

I nflammatory bowel disease (IBD)5 encompasses two distinctchronic inflammatory disease of the intestine in humans: ul-cerative colitis and Crohn’s disease. These are debilitating

diseases of unknown etiology and for which there exist only lim-ited therapeutic options (1, 2). Recently, there has been growingevidence that IBD results from abnormal immune responses tonormal gut bacterial flora. The human intestinal tract contains hun-dreds of different bacterial species that are largely tolerated by thehost. It is hypothesized that this tolerance to enteric bacteria existsbecause of the effectiveness of the epithelial barrier in separatingthe mucosal immune system and enteric Ags and/or because ofactive or indirect suppression of mucosal immune responses tosuch Ags. There is some evidence that patients with IBD can havestrong immune responses to enteric flora, although these responsesare absent in people without IBD (3, 4). In addition, allelic vari-ation in the gene for NOD2, a pattern recognition receptor (PRR)that binds muramyl dipeptide, has been associated with suscepti-bility to Crohn’s disease (5–7). Although no single bacterial spe-

cies has been associated with the development of either Crohn’sdisease or ulcerative colitis, probiotic therapies, which aim to de-crease disease severity by changing the flora of the gut, are cur-rently being tested in various clinical trials and have shown someefficacy (8). The strongest evidence for the role of bacteria in thedevelopment of IBD comes from the myriad of mouse models ofcolonic inflammation. Many of these murine models clearly showa dependence on the presence of enteric bacteria for the inflam-matory process: when rendered germfree, these mice do not de-velop colitis (9). Therefore, it may be possible to ameliorate IBDby blocking the ability of the mucosal immune system from re-sponding to bacterial Ags.

The innate immune system recognizes the presence of specificbacterial Ags through pattern recognition receptors (PRR). TLR4is one of an extensive family of PRR that have been found inDrosophila melanogaster and mammals, and compelling researchhas shown that LPS, which is the major component of the outermembrane of Gram-negative bacteria, binds to TLR4 (10–12). Therecognition of LPS requires a complex interaction of LPS withTLR4, MD-2, and CD14 (13–15). The triggering of TLR4R com-plex signaling by LPS results in a cascade of events that leads tothe secretion of proinflammatory mediators from monocytes anddendritic cells, which ultimately leads to the activation of the ac-quired immune response (16, 17). In addition to LPS, there isevidence that other ligands may bind to the TLR4R complex, par-ticularly molecules such as hyaluronic acid and heparan sulfatethat are present during active inflammation (18). Thus, signalingthrough the TLR4R complex actively contributes to the develop-ment of inflammation and may help to maintain an ongoing in-flammatory response. Therefore, in conditions of uncontrolled in-flammation, blocking TLR4 signaling may be beneficial.

CRX-526 is a synthetic lipid A mimetic molecule, also knownas an aminoalkyl-glucosaminide-phosphate (AGP) (Refs. 19 and20; see Fig. 1). Lipid A is the active component of LPS that bindsto TLR4, and AGP were developed to study the importance ofstructure on the function of lipid A (21, 22). We have establishedpreviously that CRX-526, unlike lipid A or other AGP, does not

*Corixa Corporation, Seattle, WA 98101; †Department of Immunology, The ScrippsResearch Institute, La Jolla, CA 92037; ‡Corixa Corporation, Hamilton, MT 59840;§Infectious Disease Research Institute, Seattle, WA 98104; and ¶Washington NationalPrimate Research Center, University of Washington, Seattle, WA 98195

Received for publication April 22, 2004. Accepted for publication March 8, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Corixa Corporation and by Contract N66001-01-C-8007 (to D.H.P.) from the Defense Advanced Research Projects Agency.2 Current address: CombiMatrix Corporation, Mukilteo, WA 98275.3 Address correspondence and reprint requests to Dr. Steven P. Fling, Corixa Corpo-ration, 1900 9th Avenue, Seattle, WA 98101. E-mail address: steve.fling@corixa.com4 Current address: Dendreon Corporation, Seattle, WA 98121.5 Abbreviations used in this paper: IBD, inflammatory bowel disease; PRR, patternrecognition receptor; AGP, aminoalkyl-glucosaminide-phosphate; SAC, secondaryfatty acyl chain; DSS, dextran sodium sulfate; MPL, monophosphoryl lipid A;MDR1a, multidrug resistance gene 1a; AF, aqueous formulation containing 216�g/ml dipalmitoylphosphatidyl choline; TeoA, formulation containing triethanol-amine; DAI, disease activity index; KO, knockout.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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stimulate cytokine production or other gene expression in humanperipheral blood monocytes in vitro or induce an inflammatoryresponse in vivo and that this lack of proinflammatory activity isdirectly related to the length of its secondary fatty acyl chains(SAC) (22). In the present study, we show that CRX-526 is anantagonist for the TLR4R complex and can block the proinflam-matory actions of LPS both in vitro and in vivo. This antagonistactivity directly depends on the presence of a hexanoic SAC in theleft and middle positions of the molecule. Furthermore, we wereable to demonstrate the potential importance of the TLR4R com-plex in IBD as treatment with CRX-526 successfully inhibited thedevelopment of moderate-to-severe disease in the dextran sodiumsulfate (DSS) and in the multidrug resistance gene 1a(MDR1a)-deficient mice.

Materials and MethodsCRX-526, CRX-567, CRX-568, and CRX-570

The synthesis of CRX-526 and other AGP has been described previously(19, 22). Briefly, the AGP were prepared by a highly convergent method,which allowed chemical differentiation of the hydroxyl and amino groupsand sequential introduction of the (R)-3-n-alkanoyloxytetrahexanoyl resi-dues. The AGP were purified by flash chromatography on silica gel (to�95% purity) and analyzed as a triethylammonium salt by standard ana-lytical methods. For stimulation in vitro, the AGP were formulated in watercontaining 216 �g/ml dipalmitoylphosphatidyl choline (aqueous formula-tion (AF)), 0.2% triethanolamine (TEoA, pH 7.4), or in 2% glycerol (i.v.formulation).

Animals and reagents

Female BALB/cAnN mice (5–7 wk old) were obtained from Charles RiverLaboratories; female BALB/cJ mice (5–7 wk old) and C.C3H.Tlr4Lps-d/Jmice (4–6 wk old) were obtained from The Jackson Laboratory; and fe-male MDR1a-deficient mice (5–6 wk old) were obtained from TaconicFarms. LPS from Escherichia coli 0127:B8 (Sigma-Aldrich) was recon-stituted in water at a concentration of 1 mg/ml and frozen at �20°Cuntil use.

For cytokine-specific ELISA, the following mAb pairs (BD Bio-sciences) were used: IL-6 capture no. 18871D, detection no. 18882D;IL-12 detection no. 20512D, TNF-� capture no. 18631D, and detection no.18642D. The IL-12p70 capture Ab no. 24910.1 was purchased from R&DSystems, and the MIP-1� Cytosource ELISA kit (no. CHC2204) was pur-chased from BioSource International. Murine TNF-� was determined withthe mTNF-� Quantikine kit (R&D Systems).

Human monocyte-derived macrophages stimulation with LPS

Human PBMC were isolated by Ficoll-Hypaque 1.077 (Sigma-Aldrich)centrifugation of leukapheresis product before aliquoting and freezing.Monocytes and monocyte-derived macrophages were prepared by an ad-herent step as described previously (22, 23). Phenotypic analysis of thecells revealed that �95% of the cells are CD14 positive. Stimulation ofhuman monocyte-derived macrophages in the presence of LPS and/or AGPhas been described previously in detail (22, 23). Briefly, monocyte-derivedmacrophages were exposed to CRX-526 at various concentrations. Thirtyminutes later, LPS was added to the cultures. After 6 h of stimulation, the

supernatant was removed and analyzed by ELISA for the presence ofTNF-�. RNA was isolated from adherent cells as previously described forthe generation of cDNA for microarray analysis (22).

Inhibition of LPS-induced cytokine production in monocytes

Human monocytes were generated from 4 � 106 PBMC by an adherentstep and were cultured for 48 h in RPMI 1640 medium containing 2%human AB serum. For experiments determining IL-12p70 production, theculture medium was supplemented with IFN-� (1000 U/ml) for the last24 h. After incubating with CRX-526 for 30 min, monocytes were stimu-lated with 100 ng/ml E. coli LPS. Cytokines and MIP-1� were determinedin supernatants taken after 18 h. Quantification of IL-6, IL-12p70, andTNF-� production was done by cytokine-specific ELISA as described pre-viously (23). The working sensitivity of all ELISA was shown to be �10pg/ml. MIP-1� and murine TNF-� levels were determined by using ELISAkits, according to the manufacturer’s protocol. Cytokines were not detect-able in the absence of stimulation.

Microarray analysis

A detailed description of the microarray analysis can be found elsewhere (22).Briefly, �1200 PCR products and controls were spotted onto 42 CMT-GAPS2-coated slides in triplicate (Corning Glass) using an Affymetrix 417arrayer (Affymetrix). Fluorescently labeled probes were generated from 1.5 �gof cDNA amplified as described above. The Cy3 (treated)- or Cy5 (untreated)-labeled probes were generated using the amino-allyl method as previouslydescribed (22) and included in the microarray resources section of the Instituteof Genomic Research web site: �http://pga.tigr.org/PDF/BiotechniquesCook-book_II.pdf�. Hybridizations and posthybridization washes were conducted asdescribed by the slide manufacturer (Corning Glass) for DMSO arrays usinga formamide hybridization buffer. Slides were scanned using an Affymetrix418 microarray scanner and saved as 16-bit TIFF files (Affymetrix). The Cy3and Cy5 images for each array were overlaid, gridded, and quantified usingImagene software (Biodiscovery). Hybridization data quality was evaluatedusing the mean signal-to-noise ratios for all cDNA spots, the mean signal-to-noise ratios for control spots, treated-to-untreated ratios for control andspike-in spots, and spot morphology. Normalization and additional data anal-ysis was performed using GeneSpring (Silicon Genetics). The hybridizationintensity of each spot was normalized to the median intensity of all noncontrolspots on the array.

HeLa cell transfection and reporter assay

A previously described HeLa transient transfection system was used to testthe ability of CRX-526, CRX-567, CRX-568, and CRX-570 to act as an-tagonist/agonist for the TLR4-MD2-CD14R complex (14, 22). Briefly,HeLa cells were transfected with 50 ng of IL-8 promoter-derived luciferasereporter and 10 ng each of TLR4, MD-2, and CD14 plasmids. Eighteenhours after transfection, cells were stimulated with varying concentrationsof AGP in the presence of vehicle or 100 ng/ml LPS for 6 h, lysed, and thenanalyzed for luciferase and �-galactosidase activity.

DSS model of intestinal inflammation

Two separate protocols were used to cause DSS-induced intestinal inflam-mation, depending on the strain of mice and the lot of DSS (ICN Biomedi-cals) used. For experiment no. 1 (see Fig. 1), BALB/cAnN females weregiven 4.0% DSS (lot no. 4379F) in their water ad libitum starting on day0, with all mice sacrificed on day 7. Negative control animals remained onstandard facility water. Starting on day 0, mice were given s.c. injectionsof either AF vehicle or varying doses of CRX-526-AF every other day for

FIGURE 1. The chemical structures of MPL andCRX-526.

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a total of four doses (n � 5/group). For experiment no. 2 and Fig. 5,BALB/cJ females and C.C3H Tlr4Lps-d/J mice were given 3.0% DSS (lotno. 8296F) in their drinking water from days 0 to 7 and were s.c. injecteddaily with either AF-vehicle or 50 �g of CRX-526-AF from days �3through 6 for a total of nine doses (n � 10/group). Disease activity index(DAI), a clinical score that factors in weight loss, changes in stool consis-tency, and the presence of blood in the stool was assessed by a previouslypublished method (24). Paraffin-embedded sections of the large intestines,prepared as “Swiss rolls” (25), were stained with H&E, and histologicalscoring of disease in the cecums and colons was determined in a blindedfashion by a certified veterinary pathologist (H. Bielefeldt-Ohmann).Briefly, the cecum, proximal colon, mid-colon, distal colon, and rectumwere assessed for loss of mucosal architecture (0–4), the presence of mu-coid crypt cysts (0–4), goblet cell depletion (0–4), erosion (0–4), edema(0–4), mononuclear cell infiltration (0–4), polymorphonucleocyte and/oreosinophil infiltration (0–4), and transmural inflammation (0–4). To ob-tain a total score for each mouse, the total scores for each segment wereadded together; the maximal possible score is 160.

Treatment and assessment of colitis in MDR1a-deficient mice

Starting at 5–6 wk of age, MDR1a-deficient female mice were injected s.c.on a weekly basis with 50 �g (experiment no. 1) or 100 �g (experiment no.2) of CRX-526-TeoA or with an equal volume of TeoA alone (vehicletreated). Treatment continued for 5–6 wk, and then, mice were sacrificedand the tissues prepared as described above. Histological scoring of diseasein the large intestine was performed in a blinded fashion by Dr. H.Bielefeldt-Ohmann, using a previously described scoring system (25).Briefly, the cecum, proximal colon, distal colon, and rectum from eachmouse were scored on severity of mucosal epithelial changes (0–4), degreeof inflammation (0–4), the percentage of intestinal segment affected in anymanner (extent score A), and the percentage of intestinal segment affectedby the most severe score (�2) given for inflammation or mucosa (extentscore B). The segment score was determined by summing the severityscores (mucosal score � inflammation score � extent score A � extent

score B), and then, the segment scores were added together to reach a totalscore for each mouse, with a maximum possible score of 64.

Statistics

For statistical analyses of in vivo experiments, data were analyzed by theMann-Whitney nonparametric test using Prism software (GraphPad Soft-ware). Values of p � 0.05 were deemed statistically significant.

ResultsCRX-526 inhibits the activation of human monocytes by LPS invitro

We have established previously that CRX-526, though structurallysimilar to lipid A (Fig. 1), did not by itself induce the productionof proinflammatory cytokines from human monocytes (22). Todetermine whether CRX-526 interacts with the TLR4R complex,we tested whether CRX-526 could antagonize the proinflammatoryproperties of a known TLR4 ligand, LPS. Human monocyte-de-rived macrophages were preincubated with increasing concentra-tions of CRX-526 and then exposed to various concentrations ofLPS. Preincubation of macrophages with CRX-526 resulted in aninhibition of TNF-� production after exposure to LPS (Fig. 2A). Inagreement with our previous studies, incubation of CRX-526 alonewith macrophages did not result in any TNF-� production (Ref.22; data not shown). A minimum w/w ratio of 5:1 CRX-526: LPSwas necessary to see a clearly detectable inhibition of TNF-� pro-duction; a w/w ratio of 50:1 was required to completely abolishTNF-� production in response to LPS. Preincubation of humanmonocytes with CRX-526 also resulted in inhibition of IL-6, MIP-1�, and production after exposure to LPS (Fig. 2B). Because

FIGURE 2. CRX-526 inhibits LPS stimulation of human monocytes and monocyte-derived macrophages. A, Dose dependency of CRX-526 inhibitionof LPS. TNF-� produced by human monocyte-derived macrophages exposed to 10 ng/ml LPS alone (�), LPS � 1 �g/ml (F), 5 �g/ml (Œ), or 10 �g/ml(f) CRX-526. The data of one of three separate experiments with similar results is shown. B, Inhibition of IL-6 and Mip-1�. Supernatants from monocytesincubated with CRX-526 (30 min) and then stimulated with 100 ng/ml E. coli LPS were analyzed by ELISA for IL-6 (Œ), Mip-1� (F), and TNF-� (‚).Data are presented as percent inhibition of CRX-526 treatment compared with LPS stimulation without AGP (medium). Results are shown as means SD for the two donors tested. C, CRX-526 inhibition of IFN-�-activated monocytes. Human monocytes were cultured with IFN-� (1000 U/ml for 24 h)and then incubated with CRX-526 for 30 min before stimulation with 100 ng/ml LPS in the presence IFN-�. IL-6 (Œ), IL-12p70 (f), and TNF-� (‚) weredetermined in supernatants taken after 18 h. D, Microarray analysis of changes in gene expression in human monocyte-derived macrophages stimulated withLPS in the presence or absence of CRX-526 or with CRX-526 alone.

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IFN-� activation of monocytes not only enhances the secretion ofTNF-� but also is required for the production of biological activeIL-12p70 in response to LPS (23), we determined the inhibitoryeffect of CRX-526 on the LPS-induced cytokines secretion in IFN-�-activated monocytes (Fig. 2C). In contrast to resting monocyteswhere the secretion of IL-6 and TNF-� were inhibited at similarlevels (Fig. 2B), CRX-526 had a differential inhibitory effect on thecytokine secretion of IFN-�-activated monocytes in response toLPS (Fig. 2C). A 10:1 w/w ratio of CRX-526 to LPS inhibitedIL-12p70 and TNF-� production by 79 and 57%, respectively, buthad no effect on the LPS-induced IL-6 production. In a murinesystem, CRX-526 inhibited the LPS-induced production of IL-6,IL-12p70, and TNF-� by murine splenic dendritic cells (data notshown), indicating that CRX-526 inhibits LPS signaling througheither human or murine TLR4R complexes. Antagonism of LPSsignaling by CRX-526 in human dendritic cells has also been dem-onstrated (P. Probst, unpublished observations).

To confirm that CRX-526 is a pure antagonist and does notinduce any gene transcription by signaling through the TLR4Rcomplex, we used a custom microarray containing �300 inflam-matory gene targets arranged in triplicate to look at mRNA ex-pression in human monocytes after exposure to CRX-526 aloneand/or in the presence of LPS (22). Exposure of monocytes to LPSalone (Fig. 2D, left) stimulated the expression of �60 array ele-ments �2-fold, while exposure to CRX-526 alone (Fig. 2D, right)did not induce expression of any genes found on the microarray.Furthermore, pretreatment of monocytes with increasing amountsof CRX-526 before exposure to LPS completely suppressed allLPS-induced gene transcription (Fig. 2D). NF-�B is known to beinvolved in the transcriptional regulation of many of the elementson this microarray (22). The level of inhibition by CRX-526 ofrepresentative NF-�B-regulated genes is summarized in Table I.Thus, CRX-526 can act as an antagonist for the TLR4R complexand can inhibit the ability of LPS to signal through the TLR4Rcomplex.

The antagonistic activity of CRX-526 is dependent on SAClength

The structure of CRX-526 differs significantly from monophos-phoryl lipid A (MPL) and other TLR4-agonist AGP in the lengthof its SAC: CRX-526 contains 3 SAC of 6 carbons in length,whereas MPL and other AGP, which signal through the TLR4Rcomplex, contain SAC of �10 carbons in length (Ref. 22; Fig. 1).

To confirm whether hexanoic SAC are a requirement for the an-tagonistic activity of CRX-526, we synthesized molecules that arevariants of CRX-526, containing either hexanoic or decanoic SACat three different positions (Fig. 3). CRX-567 differs from CRX-526 by only a decanoic SAC at the right position, whereas CRX-568 has a decanoic SAC in the middle position, and CRX-570 hasa decanoic SAC at the left position (compare Fig. 1 vs Fig. 3).These molecules were then tested for their ability to inhibit LPSsignaling through the TLR4R complex. Using a previously de-scribed model system (14), HeLa cells were transfected transientlywith DNA encoding TLR4, MD-2, and CD14, along with humanIL-8 promoter-driver luciferase reporter plasmid and �-galactosi-dase vector. These transfected cells were stimulated with increas-ing concentrations of either AGP alone (�) or AGP � 100 ng/mlLPS (f), and luciferase activity was monitored (Fig. 3). CRX-526inhibited, in a dose-dependent manner, the ability of LPS to signalthrough the TLR4/MD2/CD14 complex. LPS was not able to stim-ulate HeLa cells that were transfected with a control vector nor didCRX-526 alone induce a signal in the TLR4/MD2/CD14-trans-fected cells (Ref. 14; Fig. 3). CRX-567 has antagonistic activity forthe TLR4/MD2/CD14R complex similar to CRX-526 (Fig. 3).However, CRX-568 has less antagonistic activity compared withCRX-526 or CRX-567 and even some partial agonist activity.CRX-570 has almost no antagonistic activity and significant ago-nistic activity. These findings show that a shortened (hexanoic)SAC on the left and middle position of the molecule are requiredfor the inhibition of LPS signaling through the TLR4/MD2/CD14complex by CRX-526.

CRX-526 can inhibit LPS-induced TNF-� release in vivo

Our data clearly show that CRX-526 can inhibit LPS signalingthrough the TLR4R complex in vitro. To test whether CRX-526can also inhibit LPS activity in vivo, we injected LPS i.v. in thepresence or absence of CRX-526 into mice and 1 h later measuredTNF-� in the serum. We were able to detect significant amounts ofTNF-� in the serum of mice given as little as 5 ng of LPS (data notshown). Therefore, we injected mice with either 5 ng of LPS aloneor with increasing amounts of CRX-526. Significant inhibitionof TNF-� release was seen with as little as 80 ng of CRX-526(data not shown), and background levels of TNF-� werereached in the presence of 10 �g of CRX-526 (Fig. 4). Thesedata confirm that CRX-526 acts as an antagonist for LPS in vivoas well as in vitro.

Table I. CRX-526 suppression of LPS-induced gene transcription for representative NF-�B-regulated geneelementsa

Element Gene Description

Treatment (expression level)b

LPS LPS/CRX526

SCYB10 Small inducible cytokine subfamily B member 10 29.8 0.96IL-2R� IL-2 receptor, � (IL2RA) 12.4 0.04PTX3; TSG-14 Pentraxin-related gene 8.14 0.41PTGS2 Prostaglandin-endoperoxide synthase 2 (COX-2) 7.09 0.43TAP1 Peptide transporter 6.27 1.53IL-1RA IL-1 receptor antagonist 5.89 1.00GRO1 GRO1 oncogene (SCYB1) 5.33 1.88IL-1B IL-1, � 3.77 2.15CD48 Pan-leukocyte antigen 3.13 1.04NFKBIA Nuclear factor of � light polypeptide gene enhancer 2.75 1.27CD95 Apo-1 Fas 2.41 0.61IL-6 IL-6 2.18 0.26

a Values were determined from the analysis shown in Fig. 2 for specific miroarray elements that have been identified byliterature searches as having NF-�B-regulated transcription.

b Hybridization intensities were quantified using Imagene software and normalized and analyzed using Genespring software.Expression levels (-fold scale) are expressed for treated relative to untreated controls.

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CRX-526 can inhibit the development of disease in two differentmouse models of colitis

Because LPS is the major inflammatory portion of the cell mem-brane of Gram-negative bacteria, we hypothesized that CRX-526may be able to inhibit the ability of Gram-negative bacteria tocause or perpetrate the inflammation seen in IBD. Therefore, wetested the ability of CRX-526 to inhibit the development of diseasein two different mouse models of colonic inflammation: dextransodium sulfate-induced colitis, and MDR1a-deficient mice.

Exposure to DSS in drinking water causes an acute colitis inmice due to damage to the epithelium layer. DSS-exposed animalsshow distinct clinical signs, including weight loss, changes in stoolconsistency, and blood in the stool and/or gross intestinal bleeding,

that correlate directly with histological changes of the large intes-tine (24). Mice that are deficient in TLR4 expression do developacute colitis when exposed to DSS. However, Lange et al. (26)have observed that TLR4-mutant mouse strains, such as C3H/HeJand C57BL/10ScN, show delayed onset of intestinal bleedingcompared with strain-matched TLR4-wild-type mice and show in-creased survival upon exposure to DSS for �10 days. Their dataindicate that TLR4 may play a minor, but detectable, role in DSS-induced colitis. Therefore, we exposed BALB/c females to DSSfor 7 days and simultaneously treated the mice with CRX-526.Mice were assessed for clinical signs of colitis (DAI) (24) and forhistological changes in the large intestine on day 7. As shown inFig. 5, exposure to DSS caused the development of significantclinical and histological changes in these mice. Furthermore, inagreement with previous studies, DAI correlated with the histo-logical score. Cotreatment with CRX-526 significantly decreasedthe histological score at all doses tested but decreased DAI only atthe 10- and 50-�g doses (experiment no. 1, Fig. 5A). These datashow that blocking TLR4R complex signaling can decrease dis-ease severity in an acute model of colitis and that a decrease inDAI is a rigorous measure of a decrease in disease severity in ourmodel system. A repeat experiment confirmed the ability of 50 �gof CRX-526 to significantly decrease the DAI score in DSS-ex-posed mice (experiment no. 2, Fig. 5B). To confirm that our resultswere due to inhibition of signaling through the TLR4R complex,we compared the ability of CRX-526 to affect disease severity inTLR4-wild-type (BALB/cJ) and TLR4-mutant (C.C3H.Tlr4Lps-d)mice. Both TLR4-wild-type and TLR4-mutant mice developedacute disease when exposed to DSS, although the TLR4-mutantmice had a significantly lower DAI (mean DAI of 1.9 0.4 and1.4 0.2, respectively, for TLR4-wild-type and TLR4-mutantmice; p � 0.004; Fig. 6). Treatment with 50 �g of CRX-526 bys.c. injection significantly decreased the DAI score in TLR4-wild-type but not TLR4-mutant mice (Fig. 6). These data confirm that

FIGURE 4. CRX-526 inhibits LPS toxicity in vivo. Mice received 5 ngof LPS alone (p) or 5 ng of LPS plus 0, 2, 10, or 50 �g of CRX-526 byi.v. injection (n � 3/group). Negative control mice received only PBS.Error bars indicate SD. Detection of serum TNF-� as described in Mate-rials and Methods. One of three separate experiments with similar resultsis shown.

FIGURE 3. The structure of CRX-526 is critical for its TLR4 antagonistactivity. HeLa cells that had beentransfected with TLR4/MD-2/CD14were incubated in the presence of dif-ferent AGP and then stimulated with100 ng/ml LPS (f) or vehicle (�).The chemical structure of each AGPis shown directly below the corre-sponding graph, with the carbonlength of the fatty acyl chains indi-cated in parentheses. The data of oneof three independent experimentswith similar results is shown.

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the decreased disease severity seen in CRX-526-treated animals isdue to blocking of TLR4R complex signaling. Interestingly, treat-ment of TLR4-wild-type mice with CRX-526 brought the DAIscore to a level similar to that of the TLR4-mutant mice (meanDAI of 1.4 0.3 for TLR4-mutant mice and 1.3 0.8 for CRX-526-treated TLR4-wild-type mice), suggesting that at the 50-�gdose we obtained an almost complete inhibition of TLR4R com-plex signaling (Fig. 6).

As DSS exposure induces acute colitis, we wanted to test theefficacy CRX-526 in a chronic model of colitis. MDR1a-deficientmice spontaneously develop chronic inflammation of the colonwith age. As the colitis seen in MDR1a-deficient mice can beprevented by treatment with oral antibiotics, this model is clearlydependent on the presence of bacterial Ags in the gut (27). Fur-thermore, evidence suggests that MDR1a-deficient mice developcolitis due to leakiness in the epithelial barrier of the intestine,which may allow bacterial Ags to stimulate the immune system(27). Therefore, we hypothesized that Gram-negative bacteria maypotentially play a role in the development of colitis in MDR1a-deficient mice and that inhibition of signaling through the TLR4Rcomplex could decrease disease severity. We injected s.c. 5- to7-wk-old MDR1a-deficient female mice, which were free of anyclinical signs of colitis, with 50 �g (experiment no. 1) or 100 �g

(experiment no. 2) of CRX-526 or with vehicle control for 5 wkand then sacrificed the mice 1 wk after the last dose. The cecum,colon, and rectum of each animal were examined histologically forthe extent and severity of inflammation. As shown in Fig. 7 (datapooled from experiment nos. 1 and 2), the majority of vehicle-treated animals (Fig. 7B, iii and iv) had developed moderate tosevere colitis at this time point (mean histological score � 32.3).However, the majority of the mice that had been given CRX-526had mild or no disease (Fig. 7B, i and ii) (mean histologicalscore � 16.3; p � 0.0565). These data demonstrate that theTLR4R complex is at least partially involved in the developmentof colitis in MDR1a-deficient mice and that blocking TLR4R com-plex signaling can prevent the development of moderate-to-severedisease in this model.

DiscussionWe have shown that CRX-526 is an antagonist for the TLR4Rcomplex and prevents the development of colonic inflammation inmice. CRX-526 blocks the ability of LPS to trigger proinflamma-tory responses from human monocytes both in vitro and in vivo.This antagonistic activity of CRX-526 is strictly dependent on themolecule’s structure: changes in the length of SAC led to a de-crease in antagonistic activity and an increase in agonist activity

FIGURE 5. CRX-526 inhibits the development of moderate-to-severe clinical and histological disease in the DSS model of colitis. A, Experiment no.1: BALB/cAnN females were exposed to DSS for 7 days and treated every other day with various doses of CRX-526 or vehicle control. n � 5/group. Uppergraph shows DAI (clinical score), and lower graph shows histological score for the large intestine. See Materials and Methods for details on treatment withDSS, CRX-526, DAI, and histological scoring. B, Experiment no. 2: BALB/cJ females were exposed to DSS for 7 days and treated daily with CRX-526or vehicle control. See Materials and Methods for details on treatment with DSS, CRX-526, and DAI scoring; histological scores were not determined. n �10/group. A and B, Water control groups were not exposed to DSS. Error bars indicate SD, and p values for CRX-526 treatment groups vs vehicle treatmentgroups are indicated in parentheses.

FIGURE 6. CRX-526 inhibits thedevelopment of clinical disease inwild-type, but not TLR4-deficient,mice exposed to DSS. Treatment ofBALB/cJ and C.C3H Tlr4Lps-d/J micewith DSS and CRX-526 and assess-ment of DAI as described in Materi-als and Methods. n � 10/group. Thewater control group was not exposedto DSS. Error bars indicate SD; valuesof p for CRX-526 treatment group vsvehicle treatment group indicated inparentheses.

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for the TLR4R complex. Most importantly, treatment with CRX-526 was able to block the development of moderate-to-severe co-litis in two independent mouse models. In both DSS-induced co-litis and MDR1a-deficient mice, exposure to enteric Ags ismediated via defects in the epithelial barrier of the large intestine.Although not all enteric bacteria are Gram negative, these resultssuggest that signaling through the TLR4R complex may be criticalfor the development of colonic inflammation because LPS and/orother TLR4 ligands are important factors in the induction of colitisin these models. Thus, a TLR4R antagonist, such as CRX-526,may be an effective treatment for IBD as well other chronic in-flammatory diseases where TLR4 plays a significant role.

Little is known about the role of TLR4 in most of the mousemodels of colitis. In the DSS model, evidence suggests that TLR4may play a partial role in the development of severe disease, atleast in certain mouse strains (26). Our results confirm these find-ings in this DSS model. However, a genetic variant of the TLR4-mutant C3H/HeJ mice, C3H/HeJ-Bir mice, is susceptible to thespontaneous development of chronic colitis (28), suggesting thatgenetic factors can influence the relative importance of TLR4 inthe development of colitis. A role for TLR4 in the development ofchronic colitis has been demonstrated clearly in one mouse model

of colitis. Mice that have a myeloid-specific deletion of Stat3 (con-ditional Stat3 knockout (KO) mice) have enhanced Th1 responsesand develop chronic colitis, probably due to the inability of my-eloid cells to respond to IL-10 (29). Kobayashi et al. (30) havedemonstrated that the chronic colitis seen in conditional Stat3 KOmice is dependent on the presence of TLR4, IL-12p40, and T cellsbecause conditional Stat3 KO mice that are also deficient in any ofthese molecules or cells do not develop colitis. They hypothesizethat enteric bacterial products signaling through the TLR4R com-plex induce the production of IL-12 and/or IL-23 that leads to thedevelopment of Th1 cells, which drive the intestinal inflammationseen in this model (30). Our observations confirm that blockingTLR4R complex signaling can result in decreased intestinal in-flammation in another model of colitis that is characterized by thepresence of Th1-type T cells, MDR1a-deficient mice (27).

There is increasing evidence in the literature that LPS is not theonly ligand for the TLR4R complex. Heparan sulfate, the extra-domain-A of fibronectin, hyaluronic acid, fibrinogen, and Strepto-coccus pneumoniae-derived pneumolysin, has been shown to sig-nal through the TLR4R complex (18). It is possible that anantagonist for LPS that binds to the TLR4R complex may be anantagonist for other TLR4 ligands. Therefore, by treating with aTLR4 antagonist such as CRX-526, one has the potential of block-ing more than LPS-induced inflammatory responses. Currently,studies are ongoing to test the ability of CRX-526 to antagonizeother TLR4 ligands. If CRX-526 can block multiple TLR4 ligands,this could explain why TLR4 antagonism is effective in preventingan inflammatory disease as complex as the colitis seen in DSS-treated and MDR1a-deficient mice.

The exact role for TLR4 in intestinal inflammation remains con-troversial. Although most studies show a role for TLR4 in inflam-matory responses, Rakoff-Nahoum et al. (31) have demonstratedrecently an important role for TLR in the maintenance of intestinalepithelial homeostasis and protection from direct epithelial injury.Their results show that mice deficient in TLR signaling, due to adeficiency in MyD88, are more susceptible than wild-type mice tointestinal epithelial damage due to exposure to DSS or to gammairradiation. Furthermore, their data argue that constitutive signal-ing through TLR in intestinal epithelial cells results in the produc-tion of tissue protective factors, such as IL-6, KC-1, and TNF (31).Although their evidence suggests that complete inhibition of allTLR signaling may be more injurious than beneficial under cir-cumstances of acute epithelial injury, it remains possible that in-hibition of TLR4 signaling by an antagonist may be beneficialunder circumstances of chronic inflammatory disease.

Although the exact mechanism of action of CRX-526 is stillunder investigation, we hypothesize that CRX-526 binds directlyto the TLR4R complex and sterically inhibits the ability of LPSand, perhaps, other ligands to bind. We have demonstrated previ-ously that MPL and other AGP molecules do not induce cytokineproduction from cells lacking TLR4 expression, suggesting theselipid A-derived molecules are strictly ligands for TLR4 (22). Re-cently, it has been shown by Akashi et al. (15) that LPS bindsdirectly to the TLR4-MD-2 complex, while CD14 can augmentthis binding. They also tested a TLR4R complex antagonist,E5531, and showed it also bound directly to TLR4-MD-2. AsE5531 has very similar LPS-antagonist activity compared withCRX-526 (32, 33), it is possible that CRX-526 also binds directlyto a TLR4-MD-2 complex.

Clearly, additional investigation is necessary to elucidate therole of TLR4R signaling in human IBD. In normal intestinal tissue,epithelial cells express little TLR4/MD-2/CD14 and are relativelyunresponsive to LPS (34, 35). However, m-Iccl2 cells derivedfrom the crypt epithelium of the murine small intestine express

FIGURE 7. CRX-526 inhibits the development of colitis in MDR1a-deficient mice. Data is pooled from two separate experiments. A, Histo-logical scores for individual mice treated with vehicle control (f) or CRX-526 (Œ) are shown. , Mean histological score for each group. B,Representative histological sections of colons from mice treated with ei-ther: i, 50 �g of CRX-526 (no disease); ii, 50 �g of CRX-526 (mild dis-ease); iii, vehicle control (ulcerative colitis); and iv, vehicle control (severeproliferative lymphoplasmocytic colitis).

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TLR4 internally, and internalization of LPS is required for TLR4Rcomplex signaling (36). Furthermore, human patients with IBDhave increased TLR4, but not TLR2, expression on their intestinalepithelial cells and increased TLR4 and TLR2 expression on lam-ina propria monocytes (37, 38). These data confirm the expressionof TLR4 in sites of inflammation in the intestine, although it is notyet clear whether expression of TLR4 on the intestinal epitheliumis a cause and/or effect of the development of IBD. Although thedefinitive role for TLR4 in human IBD remains to be determined,the evidence we present here clearly suggest that TLR4 may be acritical therapeutic target for this disease.

AcknowledgmentsWe thank Drs. Sally Mossman, Mark Alderson, and Ken Grabstein forhelpful discussions of this manuscript and Andrea Reitsma for expert tech-nical assistance.

DisclosuresA. Mozaffarian, D. A. Johnson, D. H. Persing, P. Probst, E. Jeffery, andS. P. Fling are current employees with stock or equity interests in CorixaCorporation. A. G. Stover, R. T. Crane, R. M. Hershberg, and M. M. Fortwere previous (within the past 5 years) employees of Corixa Corporation.H. Bielefeldt-Ohmann was paid as a consultant for these studies.

References1. Sands, B. E. 2000. Therapy of inflammatory bowel disease. Gastroenterology

118:S68–S82.2. Sandborn, W. J., and S. R. Targan. 2002. Biologic therapy of inflammatory bowel

disease. Gastroenterology 122: 1592–1608.3. Duchmann, R., I. Kaiser, E. Hermann, W. Mayet, K. Ewe, and

K. H. Meyer zum Buschenfelde. 1995. Tolerance exists towards resident intes-tinal flora but is broken in active inflammatory bowel disease (IBD). Clin. Exp.Immunol. 102: 448–455.

4. Duchmann, R., M. F. Neurath, and K. H. Meyer zum Buschenfelde. 1997. Re-sponses to self and non-self intestinal microflora in health and inflammatorybowel disease. Res. Immunol. 148: 589–594.

5. Beutler, B. 2001. Autoimmunity and apoptosis: the Crohn’s connection. Immu-nity 15: 5–14.

6. Ogura, Y., D. K. Bonen, N. Inohara, D. L. Nicolae, F. F. Chen, R. Ramos,H. Britton, T. Moran, R. Karaliuskas, R. H. Duerr, et al. 2001. A frameshiftmutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411:603–606.

7. Hugot, J. P., M. Chamaillard, H. Zouali, S. Lesage, J. P. Cezard, J. Belaiche,S. Almer, C. Tysk, C. A. O’Morain, M. Gassull, et al. 2001. Association ofNOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature411: 599–603.

8. Shanahan, F. 2001. Probiotics in inflammatory bowel disease. Gut 48: 609.9. Strober, W., I. J. Fuss, and R. S. Blumberg. 2002. The immunology of mucosal

models of inflammation. Annu. Rev. Immunol. 20: 495–549.10. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu.

Rev. Immunol. 20: 197–216.11. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Im-

munol. 21: 335–376.12. Kopp, E., and R. Medzhitov. 2003. Recognition of microbial infection by Toll-

like receptors. Curr. Opin. Immunol. 15: 396–401.13. Poltorak, A., P. Ricciardi-Castagnoli, S. Citterio, and B. Beutler. 2000. Physical

contact between lipopolysaccharide and Toll-like receptor 4 revealed by geneticcomplementation. Proc. Natl. Acad. Sci. USA 97: 2163–2167.

14. da Silva, C. J., and R. J. Ulevitch. 2002. MD-2 and TLR4 N-linked glycosylationsare important for a functional lipopolysaccharide receptor. J. Biol. Chem. 277:1845–1854.

15. Akashi, S., S. Saitoh, Y. Wakabayashi, T. Kikuchi, N. Takamura, Y. Nagai,Y. Kusumoto, K. Fukase, S. Kusumoto, Y. Adachi, A. Kosugi, and K. Miyake.2003. Lipopolysaccharide interaction with cell surface Toll-like receptor4-MD-2: higher affinity than that with MD-2 or CD14. J. Exp. Med. 198:1035–1042.

16. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteinslinking innate and acquired immunity. Nat. Immunol. 2: 675–680.

17. Barton, G. M., and R. Medzhitov. 2002. Control of adaptive immune responsesby Toll-like receptors. Curr. Opin. Immunol. 14: 380–383.

18. Johnson, G. B., G. J. Brunn, and J. L. Platt. 2003. Activation of mammalianToll-like receptors by endogenous agonists. Crit. Rev. Immunol. 23: 15–44.

19. Johnson, D. A., C. G. Sowell, C. L. Johnson, M. T. Livesay, D. S. Keegan,M. J. Rhodes, J. T. Ulrich, J. R. Ward, J. L. Cantrell, and V. G. Brookshire. 1999.Synthesis and biological evaluation of a new class of vaccine adjuvants: amino-alkyl glucosaminide 4-phosphates (AGPs). Bioorg. Med. Chem. Lett. 9:2273–2278.

20. Persing, D. H., R. N. Coler, M. J. Lacy, D. A. Johnson, J. R. Baldridge,R. M. Hershberg, and S. G. Reed. 2002. Taking toll: lipid A mimetics as adju-vants and immunomodulators. Trends Microbiol. 10: S32–S37.

21. Baker, P. J., T. Hraba, C. E. Taylor, K. R. Myers, K. Takayama, N. Qureshi,P. Stuetz, S. Kusumoto, and A. Hasegawa. 1992. Structural features that influencethe ability of lipid A and its analogs to abolish expression of suppressor T cellactivity. Infect. Immun. 60: 2694–2701.

22. 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, et al. 2003.Structure-activity relationship of synthetic Toll-like receptor 4 agonists. J. Biol.Chem. 279: 4440–4449.

23. Probst, P., Y. A. Skeiky, M. Steeves, A. Gervassi, K. H. Grabstein, andS. G. Reed. 1997. A Leishmania protein that modulates interleukin (IL)-12, IL-10and tumor necrosis factor-� production and expression of B7-1 in human mono-cyte-derived antigen-presenting cells. Eur. J. Immunol. 27: 2634–2642.

24. Cooper, H. S., S. N. Murthy, R. S. Shah, and D. J. Sedergran. 1993. Clinico-pathologic study of dextran sulfate sodium experimental murine colitis. Lab.Invest. 69: 238–249.

25. Maggio-Price, L., D. Shows, K. Waggie, A. Burich, W. Zeng, S. Escobar,P. Morrissey, and J. L. Viney. 2002. Helicobacter bilis infection accelerates andH. hepaticus infection delays the development of colitis in multiple drug resis-tance-deficient (mdr1a�/�) mice. Am. J. Pathol. 160: 739–751.

26. Lange, S., D. S. Delbro, E. Jennische, and I. Mattsby-Baltzer. 1996. The role ofthe Lps gene in experimental ulcerative colitis in mice. APMIS 104: 823–833.

27. Panwala, C. M., J. C. Jones, and J. L. Viney. 1998. A novel model of inflam-matory bowel disease: mice deficient for the multiple drug resistance gene,mdr1a, spontaneously develop colitis. J. Immunol. 161: 5733–5744.

28. Elson, C. O., Y. Cong, and J. Sundberg. 2000. The C3H/HeJBir mouse model: ahigh susceptibility phenotype for colitis. Int. Rev. Immunol. 19: 63–75.

29. Takeda, K., B. E. Clausen, T. Kaisho, T. Tsujimura, N. Terada, I. Forster, andS. Akira. 1999. Enhanced Th1 activity and development of chronic enterocolitisin mice devoid of Stat3 in macrophages and neutrophils. Immunity 10: 39–49.

30. Kobayashi, M., M. N. Kweon, H. Kuwata, R. D. Schreiber, H. Kiyono,K. Takeda, and S. Akira. 2003. Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice.J. Clin. Invest. 111: 1297–1308.

31. Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, andR. Medzhitov. 2004. Recognition of commensal microflora by Toll-like receptorsis required for intestinal homeostasis. Cell 118: 229–241.

32. Abreu, M. T., P. Vora, E. Faure, L. S. Thomas, E. T. Arnold, and M. Arditi. 2001.Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinalepithelial cell protection against dysregulated proinflammatory gene expressionin response to bacterial lipopolysaccharide. J. Immunol. 167: 1609–1616.

33. Kawata, T., J. R. Bristol, D. P. Rossignol, J. R. Rose, S. Kobayashi,H. Yokohama, A. Ishibashi, W. J. Christ, K. Katayama, I. Yamatsu, and Y. Kishi.1999. E5531, a synthetic non-toxic lipid A derivative blocks the immunobiologi-cal activities of lipopolysaccharide. Br. J. Pharmacol. 127: 853–862.

34. Means, T. K., B. W. Jones, A. B. Schromm, B. A. Shurtleff, J. A. Smith, J. Keane,D. T. Golenbock, S. N. Vogel, and M. J. Fenton. 2001. Differential effects of aToll-like receptor antagonist on Mycobacterium tuberculosis-induced macro-phage responses. J. Immunol. 166: 4074–4082.

35. Smith, P. D., L. E. Smythies, M. Mosteller-Barnum, D. A. Sibley, M. W. Russell,M. Merger, M. T. Sellers, J. M. Orenstein, T. Shimada, M. F. Graham, andH. Kubagawa. 2001. Intestinal macrophages lack CD14 and CD89 and conse-quently are down-regulated for LPS- and IgA-mediated activities. J. Immunol.167: 2651–2656.

36. Hornef, M. W., B. H. Normark, A. Vandewalle, and S. Normark. 2003. Intra-cellular recognition of lipopolysaccharide by Toll-like receptor 4 in intestinalepithelial cells. J. Exp. Med. 198: 1225–1235.

37. Cario, E., and D. K. Podolsky. 2000. Differential alteration in intestinal epithelialcell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory boweldisease. Infect. Immun. 68: 7010–7017.

38. Hausmann, M., S. Kiessling, S. Mestermann, G. Webb, T. Spottl, T. Andus,J. Scholmerich, H. Herfarth, K. Ray, W. Falk, and G. Rogler. 2002. Toll-likereceptors 2 and 4 are up-regulated during intestinal inflammation. Gastroenter-ology 122: 1987–2000.

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