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Immunity Requires T Cells and Activation of Innate Humoral Humoral Immune Response to Flagellin Requires T Cells and Activation of Innate Immunity This information is current as Catherine J. Sanders, Yimin Yu, Daniel A. Moore III, Ifor R. of September 26, 2021. Williams and Andrew T. Gewirtz J Immunol 2006; 177:2810-2818; ; doi: 10.4049/jimmunol.177.5.2810 http://www.jimmunol.org/content/177/5/2810 Downloaded from References This article cites 36 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/177/5/2810.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Humoral Immune Response to Flagellin Requires T Cells and Activation of Innate Immunity1 Catherine J. Sanders, Yimin Yu, Daniel A. Moore III, Ifor R. Williams, and Andrew T. Gewirtz2 Bacterial flagellin, the primary structural component of flagella, is a dominant target of humoral immunity upon infection by enteric pathogens and in Crohn’s disease. To better understand how such responses may be regulated, we sought to define, in mice, basic mechanisms that regulate generation of flagellin-specific Igs. We observed that, in response to i.p. injection with flagellin, generation of flagellin-specific Ig required activation of innate immunity in that these responses were ablated in MyD88-deficient mice and that flagellin from Helicobacter pylori, which is known not to activate TLR5, also did not elicit Abs. Mice lacking ␣␤ T cells (TCR␤null) were completely deficient in their ability to make flagellin Abs in various contexts indicating that, in contrast to Downloaded from common belief, generation of flagellin-specific Ig is absolutely T cell dependent. In contrast to Ab responses to whole flagella (H serotyping), responses to flagellin monomers displayed only moderate serospecificity. Whereas neither oral nor rectal adminis- tration of flagellin elicited a strong serum Ab response, induction of colitis with dextran sodium sulfate resulted in a MyD88- dependent serum Ab response to endogenous flagellin, suggesting that, in an inflammatory milieu, TLR signaling promotes acquisition of Abs to intestinal flagellin. Thus, acquisition of a humoral immune response to flagellin requires activation of innate immunity, is T cell dependent, and can originate from flagellin in the intestinal tract in inflammatory conditions in the http://www.jimmunol.org/ intestine. The Journal of Immunology, 2006, 177: 2810–2818. acterial flagella have long been recognized to be one of variety of commensal and pathogenic microbes including E. coli, the major Ags of a variety of flagellated bacteria includ- Salmonella, and Clostridium spp. Purified flagellin has been shown B ing several Salmonella species, pathogenic and commen- to function as a T cell adjuvant (7, 8). Such adjuvanticity can be sal Escherichia coli strains, Pseudomonas, etc. Accordingly, sera envisioned to underlie the elicitation of humoral immune re- from hosts infected/inoculated with such pathogens display robust sponses by flagellin, although this view is at odds with the com- and relatively specific recognition of the flagella of that bacterium, monly held belief that flagella are T independent Ags. thus serving as the basis for H-Ag serotyping used to classify iso- Flagellin-specific Ig generated during Salmonella infection pro- by guest on September 26, 2021 lates of E. coli and Salmonella typhimurium. Flagella isolated from vide protection against reinfection (9); thus, flagellin, which is also such bacteria and monomeric flagellin are efficient elicitors of such a major target of T cells in primary infection (10), has been used humoral immune responses and thus the immunogenicity of flagel- in a variety of vaccine strategies (11, 12). The role of flagellin- lin has been the basis of a variety of vaccine strategies, for example specific Ig in Crohn’s disease is less clear. Flagellin-specific Ig can inserting epitopes from influenza or HIV into the flagellin mole- be envisaged to possibly protect Crohn’s disease patients from cule (1, 2). More recently, it has become appreciated that many infection by flagellated bacteria as such patients are thought to be flagellated bacteria also readily release flagellin monomers into at increased risk of intestinal infection. Conversely, because their milieu (3). Such flagellin monomers are recognized by TLR5, Crohn’s disease is thought by many to be driven by aberrant mu- thus resulting in activating innate immunity, in particular trigger- cosal immune responses against the normally commensal micro- ing a rapid induction of proinflammatory gene expression (3, 4). flora, adaptive immune responses to flagellin may drive the chronic Moreover, flagellin monomers have also been shown to be a target intestinal inflammation that characterizes this disorder. In the latter of the elevated adaptive immune response associated with Crohn’s scenario, Abs to flagellin could be envisioned to play a role in disease, a chronic intestinal inflammatory disorder driven by seem- driving the inflammation associated with Crohn’s disease or, al- ingly inappropriate immune responses to commensal intestinal mi- ternatively, the elevated levels of Abs in Crohn’s disease may sim- croflora (5, 6). Specifically, Crohn’s disease patients exhibit ele- ply reflect increased activation of flagellin-specific T cells that are vated serum IgG and IgA responses to flagellin monomers from a sufficient to drive colitis in susceptible (i.e., immunodeficient) mice (5). In light of the roles of flagellin-specific Ig in mediating outcomes of host-pathogen interactions, we sought to define some Department of Pathology, Emory University, Atlanta, GA 30322 of the basic mechanisms that regulate generation of this aspect of Received for publication January 19, 2006. Accepted for publication June 7, 2006. humoral immunity. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods Mice 1 This work was supported by the National Institutes of Health via Center Grants to Emory University (DK064399) and University of Alabama (DK64400) and by R-01 Various strains of wild-type (WT)3 mice (BALB/c, C57BL/6, and C3H/ Grant DK061417 (to A.T.G.) and via a grant from the Broad Medical Research Pro- OuJ) as well as mice carrying a null point mutation in TLR4 (C3H/HeJ), gram (to A.T.G.). 2 Address correspondence and reprint requests to Dr. Andrew T. Gewirtz, Pathology- Whitehead Research Building 105H, 615 Michael Street, Emory University, Atlanta, 3 Abbreviations used in this paper: WT, wild type; DSS, dextran sodium sulfate; DSP, GA 30322. E-mail address: [email protected] dithiobis (succinimidyl) propionate. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 2811 FIGURE 1. Systemic administra- tion of purified flagellin elicits an in- nate and adaptive immune response independent of LPS. A–C, BALB/c mice were given i.p. 100 ␮l of HBSS containing 0–100 ␮g of purified S. typhimurium flagellin (FliC). Mice were bled at 90 min and 14 days later for measure of IL-6 and flagellin-spe- cific IgG and IgA, respectively. These results are the mean of an ex- periment performed in duplicate and representative of several experi- ments. Inset in A is a Western blot (for flagellin) of serum collected Downloaded from from a mouse at various times after i.p. injection of flagellin (blot is rep- resentative of three experiments). D–F, Groups of WT (C3H/OuJ) or TLR4-deficient (C3H/HeJ) mice (n ϭ 3) were given i.p. HBSS (Ϫ)or ␮ 10 g of flagellin. Mice were bled at http://www.jimmunol.org/ 90 min and 14 days for measurement of IL-6 and flagellin-specific IgG and IgA, respectively. Results are mean Ϯ SEM. by guest on September 26, 2021 and mice with targeted deletions in TCR␤ (Tcrbtm1Mom), TCR␦ were pretreated with streptomycin (10 mg/mouse) following procedures (Tcrdtm1Mom), or both (TCR␤␦, Tcr␤dtm1Mom) were purchased from The recently described by Hardt and colleagues (15), so as to allow a more Jackson Laboratory. MyD88null mice were originally generated by Shizuo efficient colonization and yet permit the mice to survive long enough to Akira, were extensively backcrossed onto a C57BL/6 background and generate Abs to a nonattenuated Salmonella strain. Blood was obtained via maintained on standard breeder diets. All experiments were performed on retro-orbital bleeding, and serum was prepared by allowing 30 min to clot female mice 6–12 wk of age. at room temperature followed by 10 min of centrifugation at 5000 ϫ g. Reagents Native flagellin was chromatographically purified from S. typhimurium or Immunoassays E. coli F-18, and purity was verified as previously described (3, 7). S. typhimurium FliC and FljB were purified from previously described iso- Serum IL-6 was measured via a kit assay from R&D systems. To measure genic mutants of SL3201 lacking genes for FliC or FljB, respectively. product-specific (and total) Abs, microtiter plates (from Valeant Pharma- Recombinant his-tagged flagellins FliC and FlaA were prepared as previ- ceuticals) were coated with flagellin (100 ng/well), OVA (2 ␮g/well), or E.
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