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    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. Stver,

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

    2005; 174:6416-6423; ;J Immunol

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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: 64166423.

    I nflammatory bowel disease (IBD)5 encompasses two distinct

    chronic inflammatory disease of the intestine in humans: ul-cerative colitis and Crohns 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 Crohns disease (57). Although no single bacterial spe-

    cies has been associated with the development of either Crohnsdisease 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 (1012). Therecognition of LPS requires a complex interaction of LPS withTLR4, MD-2, and CD14 (1315). 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 216g/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 oth