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The Journal of Immunology, 2006, 177: 2810-2818.
Copyright © 2006 by The American Association of Immunologists, Inc.

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

Department of Pathology, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 {alpha}beta T cells (TCRbetanull) were completely deficient in their ability to make flagellin Abs in various contexts indicating that, in contrast to 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 administration 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 intestine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bacterial flagella have long been recognized to be one of the major Ags of a variety of flagellated bacteria including several Salmonella species, pathogenic and commensal Escherichia coli strains, Pseudomonas, etc. Accordingly, sera from hosts infected/inoculated with such pathogens display robust and relatively specific recognition of the flagella of that bacterium, thus serving as the basis for H-Ag serotyping used to classify isolates of E. coli and Salmonella typhimurium. Flagella isolated from such bacteria and monomeric flagellin are efficient elicitors of such humoral immune responses and thus the immunogenicity of flagellin has been the basis of a variety of vaccine strategies, for example inserting epitopes from influenza or HIV into the flagellin molecule (1, 2). More recently, it has become appreciated that many flagellated bacteria also readily release flagellin monomers into their milieu (3). Such flagellin monomers are recognized by TLR5, thus resulting in activating innate immunity, in particular triggering a rapid induction of proinflammatory gene expression (3, 4). Moreover, flagellin monomers have also been shown to be a target of the elevated adaptive immune response associated with Crohn’s disease, a chronic intestinal inflammatory disorder driven by seemingly inappropriate immune responses to commensal intestinal microflora (5, 6). Specifically, Crohn’s disease patients exhibit elevated serum IgG and IgA responses to flagellin monomers from a variety of commensal and pathogenic microbes including E. coli, Salmonella, and Clostridium spp. Purified flagellin has been shown to function as a T cell adjuvant (7, 8). Such adjuvanticity can be envisioned to underlie the elicitation of humoral immune responses by flagellin, although this view is at odds with the commonly held belief that flagella are T independent Ags.

Flagellin-specific Ig generated during Salmonella infection provide protection against reinfection (9); thus, flagellin, which is also a major target of T cells in primary infection (10), has been used in a variety of vaccine strategies (11, 12). The role of flagellin-specific Ig in Crohn’s disease is less clear. Flagellin-specific Ig can be envisaged to possibly protect Crohn’s disease patients from infection by flagellated bacteria as such patients are thought to be at increased risk of intestinal infection. Conversely, because Crohn’s disease is thought by many to be driven by aberrant mucosal immune responses against the normally commensal microflora, adaptive immune responses to flagellin may drive the chronic intestinal inflammation that characterizes this disorder. In the latter scenario, Abs to flagellin could be envisioned to play a role in driving the inflammation associated with Crohn’s disease or, alternatively, the elevated levels of Abs in Crohn’s disease may simply reflect increased activation of flagellin-specific T cells that are 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 of the basic mechanisms that regulate generation of this aspect of humoral immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Various strains of wild-type (WT)3 mice (BALB/c, C57BL/6, and C3H/OuJ) as well as mice carrying a null point mutation in TLR4 (C3H/HeJ), and mice with targeted deletions in TCRbeta (Tcrbtm1Mom), TCR{delta} (Tcrdtm1Mom), or both (TCRbeta{delta}, Tcrbetadtm1Mom) were purchased from The Jackson Laboratory. MyD88null mice were originally generated by Shizuo Akira, were extensively backcrossed onto a C57BL/6 background and maintained on standard breeder diets. All experiments were performed on female mice 6–12 wk of age.

Reagents

Native flagellin was chromatographically purified from S. typhimurium or E. coli F-18, and purity was verified as previously described (3, 7). S. typhimurium FliC and FljB were purified from previously described isogenic mutants of SL3201 lacking genes for FliC or FljB, respectively. Recombinant his-tagged flagellins FliC and FlaA were prepared as previously described (13). Whole flagella, consisting of FliC, were prepared from Sl3201 FljB– and subsequently cross-linked with dithiobis (succinimidyl)propionate (DSP) as previously described (14). Recombinant clostridia-like flagellin, F2, a gift from Charles Elson and Yingzhi Kong (University of Alabama, Birmingham, AL) was prepared as previously described (5). Carrier-free murine rTNF-{alpha} was purchased from R&D Systems. S. typhimurium LPS (ultrapure), OVA (grade VI), and all other chemicals were purchased from Sigma-Aldrich except for dextran sodium sulfate (DSS), which was from Valeant Pharmaceuticals.

Inoculations

All experiments used three to six mice per condition. In cases where five or more mice were used per condition, up to one mouse per condition was considered an outlier, and subsequently its result excluded, if it exhibited a response of <1% of all other mice in that condition. Intraperitoneal injections were done with total volumes of 100 µl. Rectal administrations were performed with flexible No. 4 French catheters inserted 3–4 cm into the distal colon. Oral gavage was performed in 200-µl volumes using a 22-gauge round-tipped needle inserted ~2 cm into the throat (just beyond the point of gag reflex). For oral gavaging of live S. typhimurium, mice were pretreated with streptomycin (10 mg/mouse) following procedures recently described by Hardt and colleagues (15), so as to allow a more efficient colonization and yet permit the mice to survive long enough to generate Abs to a nonattenuated Salmonella strain. Blood was obtained via retro-orbital bleeding, and serum was prepared by allowing 30 min to clot at room temperature followed by 10 min of centrifugation at 5000 x g.

Immunoassays

Serum IL-6 was measured via a kit assay from R&D systems. To measure product-specific (and total) Abs, microtiter plates (from Valeant Pharmaceuticals) were coated with flagellin (100 ng/well), OVA (2 µg/well), or E. coli (LPS 10 µg/well) applied overnight in 0.1 M NaHCO3 (pH 9.6). After overnight coating, mouse sera was diluted from 1/100 to 1/100,000 in ELISA wash buffer (HBSS with 0.5% goat serum and 0.1% Tween 20) and applied to coated plates. After 1 h of incubation, product-specific Ig was detected using anti-mouse IgG (or IgM)-HRP (1/1000; Amersham) or a two-step method of anti-mouse IgA-biotin (1/5000; Southern Biotechnology) followed by avidin-peroxidase (1/10,000, Jackson ImmunoResearch Laboratories). Peroxidase was then revealed via tetramethylbenzidine substrate (Kierkegaard and Perry Laboratories) and, following treatment with H2SO4, OD was read at 450 nm. The Ab titer refers to the reciprocal of the dilution of serum equivalent at OD450 to three times background (coated wells and detection Ab).

DSS colitis

DSS colitis was induced as we and others have previously described. Briefly, mice were placed on water containing 2.5 or 3.0% DSS for 7 days before being returned to regular water (16, 17). Induction of colitis was verified by observing fecal bleeding.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Purified flagellin elicits a MyD88-dependent humoral immune response

Flagellin is a major target of adaptive immunity in response to infection with flagellated pathogens and in Crohn’s disease. In light of the dependence of adaptive immunity on innate immunity, it seemed logical that the ability of flagellin to elicit adaptive immune responses was owed to its ability to activate innate immunity. We began to examine this notion by treating mice i.p. with purified flagellin (without exogenous adjuvant) and measuring serum IL-6 (at 90 min) as a general indicator of the innate response and flagellin-specific Abs at 2 wk (and various other times). Flagellin administered in this manner was rapidly maximally (<15 min) detected in the serum (observed by Western blotting; Fig. 1A, inset), indicating that this is a systemic rather than a local treatment. In contrast to healthy humans, the vast majority of whom display clearly detectable levels of flagellin-specific Abs at dilutions of 1/1000 or greater (6, 18), untreated mice displayed very low levels of Abs to E. coli or Salmonella (usually not above background signal at 1/100 dilution), thus making them quite amenable to this experimental approach. When injected i.p., at doses of 1–100 µg/mouse, flagellin elicited a dose-dependent increase in levels of serum IL-6 and flagellin-specific IgG and IgA (Fig. 1, A–C). Importantly, using a submaximal (i.e., nonsaturating) dose of flagellin, we observed identical results in C3H/OuJ and C3H/HeJ mice (Fig. 1, D–E), which carry a point mutation in their TLR4 gene that renders them unresponsive to LPS (19), indicating that our purified flagellin does not contain sufficient levels of LPS (endotoxin) to modulate these responses. The serum Ab response to flagellin was undetectable at 2 days postinjection (OD at 1/100 dilution was not above background, i.e., secondary Ab only) and robust at 14 days postinjection, indicating that it is a primary rather than a memory response (Fig. 2). The level of response from this single injection became maximal ~1 mo after flagellin administration and persisted at this level for at least another month, declining thereafter but still well above untreated mice 8 mo later (latest time measured). A second administration of flagellin 2 wk after initial inoculation resulted in a substantially increased level of flagellin-specific IgG within 3 days of treatment. For example, for BALB/c mice given 10 µg of flagellin on days 1 and 15 had levels of anti-flagellin IgG OD values: day 15, 2.0 ± 0.3, 1.2 ± 0.4, and 0.52 ±+/–0.3; and day 18, 2.5 ± 0.3, 1.7 ± 0.3, and 1.2 ± 0.2 (for serum dilutions of 1/100, 1/1,000, and 1/10,000, respectively). Thus, our inoculated mice were indeed capable of making a further Ab response to this purified protein.


Figure 1
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  		FIGURE 1. Systemic administration of purified flagellin elicits an innate and adaptive immune response independent of LPS. A–C, BALB/c mice were given i.p. 100 µl of HBSS containing 0–100 µg of purified S. typhimurium flagellin (FliC). Mice were bled at 90 min and 14 days later for measure of IL-6 and flagellin-specific IgG and IgA, respectively. These results are the mean of an experiment performed in duplicate and representative of several experiments. Inset in A is a Western blot (for flagellin) of serum collected from a mouse at various times after i.p. injection of flagellin (blot is representative of three experiments). D–F, Groups of WT (C3H/OuJ) or TLR4-deficient (C3H/HeJ) mice (n = 3) were given i.p. HBSS (–) or 10 µg of flagellin. Mice were bled at 90 min and 14 days for measurement of IL-6 and flagellin-specific IgG and IgA, respectively. Results are mean ± SEM.

 

Figure 2
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  		FIGURE 2. Kinetics of murine Ab response to flagellin. C57BL/6 mice were injected i.p. with 50 µg of flagellin. Flagellin-specific IgG was measured at indicated times in four individual mice.

 
Two approaches were utilized to discern whether the Ab response to flagellin required the innate response. First, we measured responses to flagellin (FlaA) from H. pylori that we and others have shown lacks ability to activate TLR5 (Refs. 13 , 20 , and 21 and Fig. 3). In contrast, to recombinant flagellin from S. typhimurium (FliC), administration of purified FlaA did not induce a detectable increase in serum IL-6 or specific Abs. However, when FliC and FlaA were administered with CFA, FliC and FlaA were equipotent at eliciting specific Ig, suggesting these molecules have similar levels of antigenicity but that FliC is more immunogenic than FlaA due to its ability to activate innate immunity. Next we measured responses to S. typhimurium flagellin in mice deficient in the TLR adaptor protein MyD88, which is thought to transduce all innate immune signaling in response to flagellin. MyD88 mice did not exhibit flagellin-induced serum IL-6 and were severely impaired in their ability to make Ab responses to flagellin (Fig. 4). To test whether MyD88null mice could make Abs normally under conditions expected not to require TLR signaling, we measured the Ab responses of MyD88 mice and control mice to OVA administered with TNF-{alpha} to serve as a cytokine adjuvant. Such Ab responses were similar between WT and MyD88 mice, suggesting that MyD88 mice are not deficient in ability to make Abs per se but rather that Ab responses to flagellin, and likely many Ags and/or Ag/adjuvant mixtures, require activation of innate immunity (which is often mediated by MyD88).


Figure 3
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  		FIGURE 3. H. pylori flagellin is antigenic but not immunogenic. C3H/HeJ mice (n = 3 per condition) were injected i.p. with 100 µl of HBSS containing the indicated dose (or 5 µg) of recombinant S. typhimurium or H. pylori flagellin (FliC or FlaA, respectively). Where indicated, 50 µl of CFA were added. A and B, Serum IL-6 was measured at 90 min. C, Left, Abs to FliC generated in mice inoculated with FliC, FlaA; or HBSS; right, Abs to FlaA generated in mice inoculated with FliC, FlaA, or HBSS. D, SDS-PAGE immunoblot in which serum from mice receiving FliC or FlaA was used to probe blots of FliC and FlaA to verify that the Abs measured by ELISA were largely directed at the recombinant protein rather than a potential contaminant. E, Generation of FliC and FlaA Abs in mice inoculated in presence of CFA. E is labeled analogously to C. MW, Molecular weight.

 

Figure 4
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  		FIGURE 4. MyD88 mice do not make innate or adaptive immune response to flagellin but make T-dependent Abs when a cytokine adjuvant is used. A and B, WT (C57BL/6) or MyD88null mice (n = 3) were injected i.p. with HBSS or 50 µg of S. typhimurium flagellin (FliC). Serum IL-6 and flagellin-specific IgG were measured at 90 min and 18 days, respectively. C, WT (C57BL/6) or MyD88null mice (n = 8) were injected i.p. with 50 µg of OVA ± 1.5 µg of recombinant carrier-free TNF-{alpha}. OVA-specific IgG was measured 14 days later.

 
Generation of flagellin Abs is T cell dependent

Flagella have long been considered thymus-independent Ags based largely on pioneering studies by Nossal et al. (22) demonstrating the ability of polymerized flagella to directly activate B lymphocytes in vitro. Yet, studies measuring ability of polymerized flagella to elicit flagella-specific Abs showed a substantial (~75%) diminution in athymic (nude) or thymectomized mice (23), suggesting that although T cell help may not be absolutely necessary for this response, it can play a regulatory role. However, one might expect that such flagellin monomers would lack ability to cross-link BCRs and thus not elicit Abs in the absence of T cells. However, given the recent understanding that direct activation of B cells may occur through either cross-linking of BCR or activation of TLRs and that B cells express mRNA for the flagellin receptor TLR5 (24), one could imagine that flagellin monomers might also activate B cells directly. Thus, we decided to examine the role of T cells in generating flagellin-specific Igs. We observed that mice with an engineered deletion of TCRbeta, and thus lacking all {alpha}beta T cells, displayed a complete loss of ability to make flagellin-specific IgG (Fig. 5). Further, in contrast to results previously attained with athymic mice, TCRbetanull mice also failed to make Ab responses to flagella or flagella cross-linked with DSP to prevent its depolymerization. A similar complete inability of TCRbetanull mice to make Ig to flagellin was also observed when assaying for IgM (OD450 not above background at lowest serum dilution assayed of 1/100). A similar lack of Abs to flagellin was observed in mice lacking all T cells (i.e., TCRbeta{delta}null), whereas levels of flagellin-specific IgG were similar in WT mice and mice lacking lacking only {gamma}{delta}T cells (TCR{delta}; data not shown). Mice lacking T cells displayed early increases in serum IL-6 that were indistinguishable from WT mice (Fig. 5C), suggesting that T cells are not involved in this aspect of innate recognition of this bacterial product. We next measured the Ab responses in these TCRbetanull mice in response to LPS, one of the best defined T-independent Ags. We observed that levels of specific Igs elicited by LPS were indistinguishable between WT and TCRbetanull mice, demonstrating that, as expected, TCRbetanull mice are capable of producing Abs to a T-independent Ag. Thus, in a variety of contexts, generation of flagellin-specific Igs requires {alpha}beta T cells. This conclusion is in accordance with recent observations by Honko et al. (25).


Figure 5
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  		FIGURE 5. Ab responses to flagellin are highly T cell dependent. A, Groups (n = 4–5) of WT (C57BL/6; B6) or T cell-deficient (TCRbeta–/–) mice were inoculated with HBSS or 10 µg of isolated flagella, flagella converted to monomer by heating, or flagella were cross-linked with DSP to prevent depolymerization to monomer. Serum IgG against purified flagellin monomer were measured 14 days later. Results are shown for individual mice assayed. B, Groups (n = 3) of WT (C57BL/6) or T cell-deficient (TCRbeta–/–) mice were inoculated with HBSS or 75 µg of commercially purchased, highly purified LPS. Levels of serum IgG against such LPS was measured 14 days later by ELISA. Data are the means ± SEM observed at serum dilutions of 1/100 and 1/500. C, WT (C57BL/6) or T cell-deficient (TCR{alpha}beta–/–) mice were inoculated with HBSS or the indicated dose of flagellin, and serum IL-6 was measured at 90 min by ELISA.

 
Humoral immune responses to flagellin monomers lack high serospecificity

Immune responses to flagella are considered highly specific in that antisera generated to subspecies of flagellated microbes can be used to discriminate between such bacteria (the basis of H serotyping). Conversely, that some flagellin Abs have been observed to display substantial cross-reactivity to various flagellins by Western blotting of flagellin monomers suggests that such specificity may be largely lost on flagellin monomers which would be consistent with recent studies on the structure of flagellin monomers and their assembly into flagella that showed the majority of the monomer surface is buried in the polymer and only the small portion of the molecule that is unique is exposed on the surface of the polymerized flagella (26). To quantitatively test the notion that responses to flagellin monomers might not be highly serospecific, mice were injected with one of the two flagellin isotypes from S. typhimurium (FliC or FljB) or E. coli FliC (H5 serotype) and collected serum 2 wk later. Such sera was used to probes microtiter plates to measure their levels of IgG that could recognize each of these flagellins (Fig. 6). We observed that serum from mice receiving each flagellin type exhibited a fairly similar ability to recognize the flagellin they had received as well as the two types they had not (Fig. 6A). Similarly, serum from mice orally infected with S. typhimurium or inoculated with flagella displayed similar ability to recognize flagellin monomers from either S. typhimurium or E. coli (Fig. 6, B and C). If, however, mice were given S. typhimurium flagella that were cross-linked with DSP to prevent depolymerization, the resulting serum Abs recognized flagellin monomers from S. typhimurium but not E. coli (Fig. 6D). That, in the absence of DSP, sera from infected and/or flagella-treated mice lacked clear ability to discriminate between Salmonella and E. coli flagellin monomers suggests that in these contexts Abs may be made more to disassembled pieces of flagella (i.e., flagellin monomers) than to the polymerized structure consistent with the recent observation that Salmonella organisms contain a specific receptor-mediated mechanism to release flagellin monomers (27).


Figure 6
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  		FIGURE 6. Ab responses measured against flagellin monomers do not display high serospecificty among E. coli (E.c.) and Salmonella (S.t.) flagellins. A, C57BL/6 mice were injected with 10 µg of flagellin purified from S. typhimurium (strains expressing only FliC or FljB) or E. coli, and serum was isolated 14 days later. Serum from mice receiving each flagellin was used to probe ELISA plates coated with all three flagellins (data are means ± SEM, n = 4) at indicated serum dilutions. B, C57BL/6 mice were treated with streptomycin and then orally infected with 109 CFU of S. typhimurium (SL3201). Results are shown for three individual mice (•, Ab to Salmonella flagellin; {circ}, Ab to E. coli flagellin. C and D, Mice were inoculated with 10 µg per mouse isolated whole flagella (C) or flagella that were cross-linked with DSP (D). Serum IgG levels were measured 14 days later at the indicated dilutions. Results are means ± SEM, n = 4–5. E, C3H/HeJ mice were injected with either 10 µg of E. coli flagellin (FliC) or clostridia-like F2 flagellin. Serum IgG specific for both FliC and F2 was measured at indicated serum dilutions. Results are means ± SEM, n = 4.

 
Although the apparent antigenic similarity that we observed between these E. coli and Salmonella flagellins is consistent with their high degree of amino acid sequence (~60% identical), some flagellins that have been defined as Ags in Crohn’s disease do not share quite such a high degree of similarity. Specifically, use of serologic expression cloning to identify humoral immune targets of a spontaneously colitic mouse strain identified some flagellins similar to clostridia flagellin and having ~30% amino acid sequence identity to flagellin from E. coli or Salmonella (5). Thus, to better gauge the degree of antigenic similarity of various flagellins, we measured whether flagellin Abs made to E. coli flagellin would also recognize recombinant clostridia-like flagellin (provided by Charles Elson and colleagues at the University of Alabama and referred to by Dr. Elson as F2 flagellin). We observed that serum from mice inoculated with E. coli flagellin could indeed recognize such clostridia-like flagellin but did so to a lesser degree than to flagellin from Salmonella consistent with their relative degrees of amino acid sequence homology. However, mice receiving the F2 flagellin did not show substantial recognition of E. coli flagellin. Thus, overall these results indicate that consistent with the knowledge that some regions of the flagellin molecule are highly conserved and some are relatively unique, use of flagellin monomers to assess immune responses may afford a broad view of levels of exposure to flagellated microbes but may not always be a highly specific means of indicating whether a host has been exposed to a specific microbial species.

Induction of colitis, but not simple mucosal exposure to flagellin, leads to robust MyD88-dependent Ab response to flagellin

The existence of a potentially large reservoir of flagellin in the intestinal tract suggests that the easily detectable levels of serum flagellin-specific Ig displayed by healthy humans and elevated levels displayed by Crohn’s disease patients may be generated in response to flagellin originating in the intestinal tract. Thus, we investigated whether administration of a bolus of flagellin to the gastrointestinal tract would elicit early cytokines or serum Ig. To address this, mice were given flagellin into the lower esophagus via a round-tipped needle or into the descending colon by a flexible catheter. We observed that mice given even the highest doses of flagellin that could be delivered by oral gavage or rectal enema did not display elevated levels of serum IL-6 (at 90 min or 3 h) or lead to detectable levels of flagellin-specific serum Igs (Fig. 7). Repeat administration of flagellin did not affect this lack of observed response, nor did we observe detectable response when attempting these experiments in C57BL/6 or C3H mice (in these instances, OD at lowest dilution assayed did not differ from background, i.e., secondary Ab only). This lack of response is in striking contrast to the recent work of Strindelius et al. (28), who observed very high systemic responses from oral administrations of similar doses of flagellin. Although we do not know the reason for this difference, our results suggest that generation of high levels of serum anti-flagellin IgG resulting from exposure to flagellin via the gastrointestinal tract may require systemic or at least extramucosal exposure to this molecule.


Figure 7
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  		FIGURE 7. Oral and rectal administration of flagellin failed to elicit detectable serum IL-6 or flagellin-specific Ig. A and B, BALB/c mice were given the indicated dose of flagellin i.p. (IP), intragastrically (IG), or intracolonically (IC) as described in Materials and Methods. Serum IL-6 was assayed at the indicated time and flagellin-specific IgG assayed 15 days later. C, Flagellin was mixed 1:1 with ethanol and administered via the indicated route (100 µl containing 50 µg of flagellin) and flagellin-specific IgG measured 14 days later. Results are means ± SEM, n = 4.

 
The lack of a serum cytokine response to mucosal administration of flagellin, together with results that, in both polarized epithelial cell lines and human colon, TLR5 is not expressed at functional levels on the apical surface (29, 30), suggest that the failure of intracolonic flagellin administration to lead to a serum Ab response might result from a simple failure of such flagellin to cross the intestinal mucosa. Thus, we next administered flagellin mucosally in the presence of 50% ethanol, which results in transient disruption of the epithelial barrier. Flagellin administered to the colon in this manner indeed resulted in a detectable levels of serum Abs to flagellin although the magnitude of the response was still well below that observed in response to flagellin delivered i.p. (Fig. 7C). We next sought to induce the intestinal mucosa into a state resembling that seen in inflammatory bowel disease. Specifically, we gave mice drinking water containing DSS, which is known to result in a robust colitis thought to be initially driven by epithelial barrier disruption. After 7 days of DSS treatment, at which time mice had bloody stools and other clinical indicators of colitis as we and others have described (17, 31), mice were returned to regular drinking water, allowing an apparent healing of their colitis. We observed that such treatment with DSS, by itself, resulted in a robust Ab response to clostridia-like F2 flagellin and a mildly elevated Ab response to E. coli flagellin (Fig. 8). We next investigated whether such DSS-induced acquisition of flagellin-specific Ig shared the dependency on MyD88 that we observed for the Ab response to purified systemically administered flagellin. We reduced the concentration of DSS used to avoid the death of MyD88 mice that can occur in this model (32). Indeed, although MyD88null mice contract DSS colitis with greater severity than WT mice (32), they failed to acquire Abs to endogenous flagellin in this colitis model. We next measured whether mucosal administration of E. coli flagellin following induction of colitis might result in a greater elevation of Abs to this protein. We observed that neither oral nor intracolonic administration of E. coli flagellin given at the height of DSS colitis (after 7 days of DSS treatment) led to a significant elevation in Abs to this protein as compared with DSS alone (OD at 1/100 and 1/1000 dilutions were: HBSS, 0.12 ± 0.4 and 0.02 ± 0.05; 50 µg of flagellin intragastrically, 0.13 ± 0.6 and 0.02 ± 0.04; and 50 µg of flagellin intracolonically, 0.16 ± 0.6 and 0.03 ± 0.05), although DSS-treated mice still responded robustly to E. coli flagellin administered i.p. (OD at 1/100 and 1/1000 dilutions were 1.8 ± 0.3 and 0.7 ± 0.2, respectively), suggesting that mimicking the endogenous mucosal response by simple administration of exogenous flagellin may not be a straightforward matter. Nonetheless, together, these results suggest that generating inflammatory conditions in the intestine, and perhaps the resulting loss of epithelial barrier function, might be sufficient to result in generation of an Ab response to endogenous bacterial flagellin and that this adaptive response requires TLR signaling.


Figure 8
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  		FIGURE 8. Induction of colitis with DSS results in a MyD88-dependent acquisition of Ab response to endogenous flagellin. A, C57BL/6 mice were bled (day 0) and then given drinking water containing 3% DSS for 7 days and serum assayed for IgG to E. coli or clostridia-like flagellin (F2) 14 days after their removal from DSS. B, C57BL/6 or MyD88 mice were given 2.5% DSS for 7 days, returned to regular water, and assayed for serum IgG to clostridia-like flagellin (F2) 15 days later.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Bacterial flagella, and its monomeric subunit flagellin, has been the focus of a number of immunologic studies because of its utility as a model system, the important role of motility in bacterial pathogenesis, and, consequently, the decision of the immune system to target this molecule. Moreover, there has recently been a new level of interest in immune responses to flagellin due to the realization that flagellin is a dominant Ag in Crohn’s disease (5). Although the immunogenicity of flagellin has long been recognized, until recently the reasons underlying its immunogenicity have largely been unknown. One potential reason that has long been offered for the targeting of flagellin by humoral immunity is that polymerized flagellin (i.e., flagella) should be able to cross-link BCRs and thus be able to directly elicit a humoral response without the need for the comparatively complex process of T cell activation. However, this explanation would not be able to explain the fact that flagellin is also the dominant T cell target upon infection by S. typhimurium in WT or B cell-deficient mice (10). Moreover, our results herein indicate that in a physiologic infection, the humoral response is likely being generated to flagellin monomers rather than polymerized flagella and that flagellin monomers would be unable to cross-link BCR. Recent work by Subramanian et al. (27) indicates that bacteria have a regulated host-induced means of secreting flagellin monomers; thus, we envisage such monomers, as well as depolymerized flagella, to be the target of this humoral immune response. Most importantly, we show that generation of Abs to flagellin monomers, flagella, or cross-linked flagella is undetectable in mice lacking T cells, suggesting that direct activation of B cells by flagella/flagellin does not occur to a significant enough extent to result in a detectable humoral immune response to this bacterial product in the absence of T cell help. Although a sense of a potential role for T cells in generating Abs to flagellin can be retrospectively gleaned from the work that Huchet and Feldman (23) performed 30 years ago, it is not entirely clear why they observed a modulatory role for T cells in this process whereas we observed an absolute requirement. Some possible reasons for this difference are: 1) they used athymic (nude or thymectomized) mice that, in contrast to TCRbeta mice, may still have some extrathymically developed {alpha}beta T cells, or perhaps even a so-called second thymus that can allow for some T cell development (33); 2) they used flagella that had been dinitrophenylated which can be envisaged to strengthen its potential ability to directly activate B cells; 3) perhaps their dinitrophenylated flagella contained a product other than flagellin that had the direct ability to activate B cells, although we have observed that flagellin mixed with LPS still failed to elicit Abs to flagellin in TCRbetanull mice (data not shown), arguing against this reasoning. Regardless of whether one or more of these potential explanations played a role, generation of Abs to flagellin/flagella is greatly influenced by, if not absolutely requires, {alpha}beta T cells.

Given that, at least in our experimental system, generation of flagellin-specific Ig is an absolutely T cell-dependent response and that flagellin functions as a T cell and MyD88-dependent adjuvant (7, 8), it seemed likely that this central TLR signaling molecule might be essential for generation of flagellin-specific Ig, and indeed we observed this was the case. Thus, consistent with one of the emerging central themes in immunology, adaptive immune responses to flagellin are dependent on innate immunity, although we are not aware of another protein that has been shown to promote adaptive responses to itself by activating innate immunity. Although a recent study has shown that generation of humoral responses to T-dependent Ags uses signaling in B cells (24), we observed herein that T-dependent responses need not always require TLR signaling in B cells, particularly if the response is driven by a cytokine such as TNF-{alpha}. Given that murine dendritic cells have been observed not to express TLR5 and not directly respond to flagellin (34), it has been suggested that the dendritic cell activation that we and others observed occurs in response to flagellin in vivo (7) is driven by flagellin-induced cytokines. Together, this indicates that the failure of MyD88 mice to make Abs in response to flagellin is not due entirely to a need for MyD88 signaling in B cells but at least in part due to a need for MyD88 for the cytokine production that may be necessary to activate dendritic cells in response to flagellin. Importantly, our results suggest that such a role for TLR signaling is true not only in response to purified flagellin but in the acquisition of anti-flagellin Abs associated with murine colitis. Although one might imagine that such acquisition of Abs to endogenous flagellin might take place in a milieu of other microbial products/adjuvants in which the activation of innate immunity by flagellin might be redundant, we have recently observed that healthy humans carrying a dominant-negative TLR5 allele display reduced levels of flagellin-specific Ig (18). Thus, we speculate that the propensity of many flagellated microbes to readily release flagellin monomers much more readily than other bacterial-derived adjuvants (e.g., endotoxin, unmethylated CpG DNA) may result in the immune system often seeing flagellin in the absence of a plethora of other bacterial products in which context the ability of flagellin to activate innate immunity is very important. Although recent work has identified a TLR5-independent and MyD88-independent pathway for recognition of flagellin (35), our results suggest that this pathway may not provide a sufficient signal 2 to generate an Ab response.

Extrapolating our results to attempt to increase understanding of how humans naturally acquire Abs to flagellin over their lifetime and how such levels might become elevated in Crohn’s disease suggests that the substantial level of serum Abs to E. coli and Salmonella flagellin that we and others have observed are present in healthy humans likely reflect levels of activation of flagellin-specific T cells, perhaps resulting from a variety of clinical and subclinical infectious and/or transient ulcerations. Whereas the first-described T cell epitope of Salmonella flagellin is somewhat specific for Salmonella (i.e., present in many Salmonella serovars but not E. coli) (10), the T cell epitopes of Salmonella flagellin targeted during primary infection have recently been defined to be in highly conserved regions of the protein common to E. coli and other common flagellins (36). This suggests that the not highly serospecific responses observed herein is likely polyclonal rather than directed toward one specific conserved epitope of flagellin, with the highly conserved portion of the flagellin monomer that mediates polymerization and is targeted by TLR5 (14) being one structural feature that might likely be so targeted. However, interestingly, when assessing Ab cross-reactivity between these typical flagellins and a more distantly related clostridia-like flagellin that was serologically cloned as a dominant Ag in murine colitis, we observed somewhat of a one-way specificity in that Abs made in response to E. coli flagellin reacted moderately with a clostridia-like flagellin but not vice versa whether the response was made to purified protein or acquired from native flora upon induction of colitis. Although the mechanisms governing such cross-reactivity remain to be determined, the lack of significant levels of Abs to E. coli flagellin in healthy laboratory mice and their highly preferential induction of responses to clostridia-like flagellin upon induction of DSS colitis suggest that flagellated E. coli may not be a major component of our mouse microflora. However, given that mice did not respond well to a bolus of intracolonically administered E. coli flagellin under a variety of conditions but could respond well to its endogenous flagellin upon induction of colitis, it is suggested that this surface may be normally be quite strongly set to be hyporesponsive to such products except when delivered under appropriate danger signals delivered with Ags in a coordinated manner. Because mucosal administration of flagellin to other surfaces results in robust responses (2, 37), intestinal hypresponsiveness to flagellin may be restricted to surfaces with large commensal bacterial loads such as the gastrointestinal tract. Such intestinal hyporesponse to flagellin may in part be mediated by both its highly selective barrier function and/or the intestinal population of regulatory T cells.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

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 Grant DK061417 (to A.T.G.) and via a grant from the Broad Medical Research Program (to A.T.G.). Back

2 Address correspondence and reprint requests to Dr. Andrew T. Gewirtz, Pathology-Whitehead Research Building 105H, 615 Michael Street, Emory University, Atlanta, GA 30322. E-mail address: agewirt{at}emory.edu Back

3 Abbreviations used in this paper: WT, wild type; DSS, dextran sodium sulfate; DSP, dithiobis (succinimidyl) propionate. Back

Received for publication January 19, 2006. Accepted for publication June 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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