The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liang, S.
Right arrow Articles by Hajishengallis, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liang, S.
Right arrow Articles by Hajishengallis, G.
The Journal of Immunology, 2007, 178: 4811-4819.
Copyright © 2007 by The American Association of Immunologists, Inc.

The A Subunit of Type IIb Enterotoxin (LT-IIb) Suppresses the Proinflammatory Potential of the B Subunit and Its Ability to Recruit and Interact with TLR21

Shuang Liang*, Min Wang*, Kathy Triantafilou{ddagger}, Martha Triantafilou{ddagger}, Hesham F. Nawar§, Michael W. Russell§, Terry D. Connell§ and George Hajishengallis2,*,{dagger}

* Center for Oral Health and Systemic Disease, Department of Periodontics, and {dagger} Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292; {ddagger} Infection and Immunity Group, School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom; and § Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York, Buffalo, NY 14214


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The type IIb heat-labile enterotoxin of Escherichia coli (LT-IIb) and its nontoxic pentameric B subunit (LT-IIb-B5) display different immunomodulatory activities, the mechanisms of which are poorly understood. We investigated mechanisms whereby the absence of the catalytically active A subunit from LT-IIb-B5 renders this molecule immunostimulatory through TLR2. LT-IIb-B5, but not LT-IIb, induced TLR2-mediated NF-{kappa}B activation and TNF-{alpha} production. These LT-IIb-B5 activities were antagonized by LT-IIb; however, inhibitors of adenylate cyclase or protein kinase A reversed this antagonism. The LT-IIb antagonistic effect is thus likely dependent upon the catalytic activity of its A subunit, which causes elevation of intracellular cAMP and activates cAMP-dependent protein kinase A. Consistent with this, a membrane-permeable cAMP analog and a cAMP-elevating agonist, but not catalytically defective point mutants of LT-IIb, mimicked the antagonistic action of wild-type LT-IIb. The mutants moreover displayed increased proinflammatory activity compared with wild-type LT-IIb. Additional mechanisms for the divergent effects on TLR2 activation by LT-IIb and LT-IIb-B5 were suggested by findings that the latter was significantly stronger in inducing lipid raft recruitment of TLR2 and interacting with this receptor. The selective use of TLR2 by LT-IIb-B5 was confirmed in an assay for IL-10, which is inducible by both LT-IIb and LT-IIb-B5 at comparable levels; TLR2-deficient macrophages failed to induce IL-10 in response to LT-IIb-B5 but not in response to LT-IIb. These differential immunomodulatory effects by LT-IIb and LT-IIb-B5 have important implications for adjuvant development and, furthermore, suggest that enterotoxic E. coli may suppress TLR-mediated innate immunity through the action of the enterotoxin A subunit.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Escherichia coli and Vibrio cholerae bacteria produce structurally related heat-labile enterotoxins that cause diarrhea in humans and animals, and can be classified into two major types based on genetic, biochemical, and immunological properties (1). The type I subfamily includes cholera toxin and E. coli enterotoxin LT-I, whereas the type II subfamily comprises the antigenically cross-reactive LT-IIa and LT-IIb, which are expressed by E. coli strains isolated from food products, animals, or humans with diarrhea (1). Both type I and type II enterotoxins display an AB5 oligomeric structure in which an enzymatically active and toxic A subunit is noncovalently linked to a pentameric ganglioside-binding (B5) subunit (2, 3) (see Fig. 1). The actual catalytic moiety is the A1 subcomponent, which is formed by proteolytic cleavage and reduction of an intrachain disulfide bond in the A polypeptide, while the A2 subcomponent at the C-terminal end of the A polypeptide is inserted noncovalently into the central pore of the ring-shaped B pentamer (2, 3). The B pentamer in itself is nontoxic but binds with high affinity to gangliosides, a heterogeneous family of glycolipids found on most cells (4), and delivers the A subunit into the cells (1). The ADP-ribosyltransferase activity of the internalized A subunit can subsequently permanently activate the Gs{alpha} component of adenylate cyclase leading to unregulated elevation of intracellular cAMP (see Fig. 1). In intoxicated gut epithelial cells, this results in massive secretion of electrolytes and water into the gut lumen, clinically manifested as diarrhea (1). In general, however, elevated intracellular cAMP mediates multiple biological effects in a variety of cell types, mainly through activation of protein kinase A (PKA)3 and the ensuing phosphorylation of transcription factors that act through cAMP-responsive elements in the promoter region of target genes (5).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 1. Model describing differential TLR2 interactions and distinct effects on cell activation by the LT-IIb holotoxin and its B pentamer, LT-IIb-B5. LT-IIb-B5 induces lipid-raft recruitment of and interacts with TLR2 and TLR1. In contrast, the holotoxin does not efficiently interact with TLR2/1 presumably due to A subunit-dependent steric hindrance. Moreover, upon internalization of the holotoxin, the ADP-ribosyltransferase activity of its A subunit activates the Gs{alpha} component of adenylate cyclase (AC). This leads to elevation of intracellular cAMP, activation of cAMP-dependent PKA, and inhibition of NF-{kappa}B-dependent transcription of cytokines like TNF-{alpha}.

 
The ability of these enterotoxins to dramatically elevate intracellular cAMP levels, in stark contrast to their catalytically inactive B pentamers, may at least partially determine why intact toxins (holotoxins) and isolated B pentamers display distinct immunomodulatory and adjuvant activities (reviewed in Ref. 6). In this context, we found that LT-IIa and LT-IIb holotoxins (but not their B pentamers) inhibit NF-{kappa}B activation and regulate cytokine induction in monocytic cells stimulated with LPS or other proinflammatory stimuli (7). Interestingly, although LT-IIa and LT-IIb are poor inducers of cytokine release, their respective B pentamers (LT-IIa-B5 and especially LT-IIb-B5) induce relatively high levels of proinflammatory cytokines in human or mouse monocytes/macrophages (7, 8). However, the capacity of LT-IIa-B5 or LT-IIb-B5 to induce NF-{kappa}B activation and proinflammatory cytokine production is antagonized by the intact holotoxins (7). These differences may have important implications not only in terms of distinct immunomodulatory or adjuvant properties (6), but also from a bacterial pathogenesis viewpoint. In this regard, early recognition events between innate immune cells and pathogen components control cellular activation or, alternatively, determine evasion or subversion of the host response (9, 10). Therefore, the ability of LT-IIa or LT-IIb to inhibit LPS-induced cellular activation and, moreover, to suppress the inherent proinflammatory potential of their own B subunits, may down-regulate innate immunity and increase the survival capacity of enterotoxic E. coli thus prolonging an infection.

In this study, we have focused on intact LT-IIb and LT-IIb-B5 for investigating the molecular mechanisms underlying their divergent effects on macrophage activation. Both molecules bind with high affinity to ganglioside GD1a (11, 12) but only the B pentamer activates TLR2-mediated NF-{kappa}B-dependent transcription (8). Whereas LT-IIb inhibits LT-IIb-B5-induced TNF-{alpha} production, LT-IIb synergizes with LT-IIb-B5 in IL-10 induction; however, neutralization of this anti-inflammatory cytokine by specific Ab only minimally affects the inhibitory effect of LT-IIb on TNF-{alpha} production (7). We have thus turned our attention to IL-10-independent mechanisms. Although the divergent biological properties of LT-IIb and LT-IIb-B5 are likely attributable to structural differences related to the presence of the A subunit in the intact holotoxin, it is uncertain whether the differences are linked to the property of the A subunit to cause elevation of intracellular cAMP or whether some other property of the A subunit is involved. To directly address this uncertainty, we used agonists or inhibitors that regulate cAMP synthesis and/or signaling and constructed catalytically defective LT-IIb mutants for determining their effects on the proinflammatory activity of LT-IIb-B5. A related puzzling question concerned the inability of LT-IIb to activate TLR2. Specifically, it has been unclear whether this holotoxin interacts physically with TLR2 but at the same time inhibits TLR2 downstream signaling by interceptive cross-talk signaling activated in parallel via different receptor(s), or whether the holotoxin fails to recruit TLR2 into lipid rafts and/or physically bind this receptor. Our comparative analysis of LT-IIb and LT-IIb-B5 regarding these issues suggests that the presence of the A subunit is associated with reduced cell activation by interfering with efficient TLR2 interactions and by inducing cAMP-mediated inhibitory effects on NF-{kappa}B-dependent transcription (summarized in Fig. 1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Enterotoxins and other reagents

The construction of recombinant plasmids encoding His-tagged versions of wild-type LT-IIb or LT-IIb-B5 has been previously described (7). Single or dual point substitution mutations (S59K and S59K/E110K, respectively) in the catalytic domain of the LT-IIb holotoxin were engineered by means of site-directed mutagenesis (QuikChange kit; Stratagene). LT-IIb and derivatives were expressed in E. coli DH5{alpha}F'Kan (Invitrogen Life Technologies) transformed with the appropriate plasmids, and the proteins were extracted from the periplasmic space using polymyxin B treatment (7, 8). The proteins were purified by means of ammonium sulfate precipitation, followed by nickel affinity chromatography and size-exclusion chromatography using a Sephacryl-100 column and an ÁKTA-FPLC system (Amersham Biosciences). Identity and purity were confirmed by SDS-PAGE, immunoblotting using specific rabbit IgG Abs, and by quantitative Limulus amebocyte lysate assay kits (BioWhittaker or Charles River Endosafe), which determined negligible endotoxic activity (<0.007 ng/µg protein). Further evidence against contamination with LPS or other heat-stable contaminants was obtained upon LT-IIb-B5 or holotoxin boiling, which destroys their biological activities (this study and Refs. 7 , 8). E. coli LPS (ultrapure grade) was purchased from InvivoGen. H89, SQ22536, indomethacin, forskolin, dibutyryl cAMP, and methyl-beta-cyclodextrin (MCD) were purchased from Sigma-Aldrich. Monoclonal Ab to TLR2 (clone TL2.1) and IgG2a isotype control were purchased from eBioscience. The reagents were used at effective concentrations determined in preliminary experiments or in previous publications.

Cell isolation and culture

Primary human monocytes were purified from peripheral blood upon centrifugation over NycoPrep 1.068 (Axis-Shield) as previously described (13). Incidental nonmonocytes were removed by magnetic depletion using a mixture of biotin-conjugated mAbs and magnetic microbeads coupled to anti-biotin mAb (Monocyte isolation kit II; Miltenyi Biotec). Purified monocytes were cultured at 37°C and 5% CO2 atmosphere, in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.05 mM 2-ME (complete RPMI 1640 medium). Complete RPMI 1640 medium was also used to culture human monocytic THP-1 cells in the American Type Culture Collection (ATCC TIB-202). Human embryonic kidney (HEK)-293 cells (ATCC CRL-1573) were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). Chinese hamster ovary (CHO)-K1 cells (ATCC CRL-9618) were maintained in Ham’s F-12 nutrient mixture (Invitrogen Life Technologies) supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Thioglycolate-elicited macrophages were isolated from the peritoneal cavity of mice deficient in TLR2 (14), TLR4 (C3H/HeJ), both TLR2 and TLR4 (15), or from wild-type control mice (C57BL/6 or C3H/HeOuJ; The Jackson Laboratory), as previously described (8, 16). The mice harboring homozygous TLR2 and TLR4 mutations were 9-fold backcrossed toward the C3H genetic background, which was donated by Dr. C. Kirschning (Technical University of Munich, Munich, Germany). Mouse primary macrophages and the mouse macrophage cell line J774A.1 (ATCC TIB-67) were cultured in complete RPMI 1640 medium. Cell viability was monitored using the CellTiter Blue assay kit (Promega). None of the experimental treatments affected cell viability compared with medium-only control treatments. Human blood collections and isolation of mouse macrophages were conducted in compliance with established federal guidelines and institutional policies.

Cellular activation assays

Cytokine induction. Human monocytes or mouse macrophages (2 x 105/well) were stimulated with LT-IIb-B5 or wild-type or mutant LT-IIb and collected supernatants were analyzed for cytokine induction using ELISA kits (eBioscience) (17). PGE2 induction was measured using a kit from R&D Systems.

NF-{kappa}B luciferase assay. Reporter assays for NF-{kappa}B activation based on inducible luciferase activity were performed as previously described (8, 17). Briefly, HEK-293 cells were transiently cotransfected with human TLR1/TLR2 (pDUO-hTLR1/TLR2; InvivoGen) and a NF-{kappa}B reporter system (comprising a firefly luciferase reporter gene controlled by five tandem repeats of NF-{kappa}B consensus sequence cloned upstream of a basic promoter (pNF-{kappa}B-Luc; Stratagene) and a Renilla luciferase transfection control (pRL-null; Promega)). Two days posttransfection, the cells were stimulated for 6 h with agonists as per experimental protocol. The Renilla and firefly luciferase activities were subsequently measured in cell lysates using the Dual-Glo luciferase reporter assay system (Promega) and a Clarity luminescence microplate reader (Bio-Tek Instruments). Luciferase activity was calculated as a ratio of firefly luciferase activity to Renilla luciferase activity, to correct for transfection efficiency. The results were then normalized to those of unstimulated control cells transfected with reporter and empty vectors, the value of which was taken as 1.

cAMP induction. Levels of cAMP in activated cell extracts were measured using a cAMP enzyme immunoassay kit, as previously described (12) following the protocol of the manufacturer (Cayman Chemicals).

Binding assays

Binding of ligands to plate-immobilized GD1a or CD14 was assessed as we have previously described (12, 18). TLR2 binding was measured using a modification of a previously published method (19). Briefly, 96-well microtiter wells were coated overnight at 4°C with 20 µg/ml purified recombinant mouse TLR2 (R&D Systems). Nonspecific binding sites were blocked with 5% (w/v) BSA in PBS for 1 h at 37°C. Wild-type or mutant LT-IIb or LT-IIb-B5 were incubated at 10 µg/ml (in PBS containing 1% BSA) for 2 h at 37°C. After washing with PBS containing 0.05% Tween 20, bound protein was detected with rabbit IgG anti-LT-IIb Ab, followed by peroxidase-conjugated goat anti-rabbit IgG (adsorbed against human or mouse IgG). The peroxidase reaction was performed using tetramethylbenzidine chromogenic substrate, and the OD signal at 450 nm was read in a microplate reader (Bio-Tek Instruments). Because recombinant TLR2 was expressed as a fusion protein with the Fc region of human IgG (R&D Systems), binding to recombinant CD14 (which is similarly fused to the Fc region of human IgG) was used as a negative control.

For cell-based binding assays, biotinylated LT-IIb-B5 or LT-IIb (1 µg/ml) were allowed to bind to empty vector- or TLR-transfected CHO-K1 cells for 15 min at 37°C, as we have previously described for other bacterial molecules (18, 20). Subsequently, the cells were washed and incubated on ice with FITC-labeled streptavidin. After washing, binding was determined by measuring cell-associated fluorescence (in relative fluorescence units) on a microplate fluorescence reader (Bio-Tek Instruments) with excitation/emission wavelength settings of 485/530 nm. Transfections of CHO-K1 cells were performed using the PolyFect transfection reagent (Qiagen) and plasmids encoding human TLR2 (pUNO-hTLR2), TLR4/MD2 (pDUO-hMD2/TLR4) or empty control vectors (InvivoGen), as we have described previously (20).

Fluorescence resonance energy transfer (FRET)

FRET is a biophysical technique that can determine sterical coassociation of fluorescently labeled molecules on the basis of measurements of nonradiative energy transfer from the excited state of a donor molecule to an appropriate acceptor (21, 22). For FRET measurements, human monocytes were cultured on microchamber culture slides (Lab-tek; Invitrogen Life Technologies). Upon stimulation for 10 min at 37°C with LT-IIb, LT-IIb-B5 (both at 1 µg/ml) or with medium only, the cells were labeled with 100 µl of a mixture of Cy3-conjugated mAb (donor) and Cy5-conjugated mAb (acceptor). The cells were rinsed twice with PBS/0.02% BSA and then fixed with 4% paraformaldehyde for 15 min. Cell fixation was necessary to prevent potential reorganization of the proteins during the course of the experiment and energy transfer determinations. Energy transfer between different receptor pairs was calculated from the increase in donor fluorescence after acceptor photobleaching, as we have previously described (17, 21, 23). The following mAbs (clones) were used for FRET: Anti-TLR1 (GD2.F4), anti-TLR2 (TL2.1) and anti-TLR4 (HTA-125) from HyCult; anti-GD1a (GD1a-2b) from AMS Biotechnology; anti-CD14 (Tük 4) and anti-MHC class I (W6/32) from Abcam. Anti-CD14 mAb, clone 26ic, was purified from hybridoma supernatant (ATCC HB246). Cholera toxin was from List Biological Laboratories and was used to label ganglioside GM1. The conjugation of Abs or other molecules to Cy3 or Cy5 was performed using kits from Amersham Biosciences.

Statistical analysis

Data were evaluated by ANOVA using the InStat v3.06 program (GraphPad Software). Where appropriate (comparison of two groups only), two-tailed t tests were conducted. The experiments described were performed at least twice for verification. Statistical differences were considered significant at the level of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Role of cAMP-dependent PKA in LT-IIb regulatory effects on cytokine production

The presence of the A subunit in the LT-IIb holotoxin is correlated with decreased proinflammatory and increased anti-inflammatory properties compared with its isolated B pentamer, LT-IIb-B5 (7). To investigate whether these differences are attributable to the catalytic activity of the A subunit to cause elevation of intracellular cAMP levels, we pretreated human monocytes with medium (control) or with SQ22536, a cAMP synthesis inhibitor targeting adenylate cyclase (24). The monocytes were then stimulated with LT-IIb-B5, LT-IIb, or a combination of the two, to determine whether SQ22536 reverses the demonstrated abilities of LT-IIb to inhibit LT-IIb-B5-induced TNF-{alpha} production and augment LT-IIb-B5-induced IL-10 production (7). In contrast to LT-IIb, LT-IIb-B5 induced TNF-{alpha} production that was inhibited in the simultaneous presence of LT-IIb (Fig. 2A). However, SQ22536 pretreatment significantly (p < 0.05) reversed the inhibitory effect of LT-IIb on LT-IIb-B5-induced TNF-{alpha} production, which now approached the levels induced by LT-IIb-B5 alone (Fig. 2A). Interestingly, LT-IIb induced a modest TNF-{alpha} response in the presence of SQ22536, whereas such response was undetectable in the presence of LT-IIb alone (Fig. 2A).


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 2. LT-IIb antagonizes cytokine induction by LT-IIb-B5 through a cAMP/PKA mechanism. Human monocytes were exposed to 2 µg/ml LT-IIb-B5, LT-IIb, or a combination of the two, with or without 30-min pretreatment with 200 µM SQ22536 (A and B) or 10 µM H89 (C and D). Induction of TNF-{alpha} and IL-10 production in culture supernatants after 16-h incubation was determined by ELISA. Results are shown as the mean ± SD (n = 3) from one of two sets of experiments that yielded similar findings. Cytokine responses in cells treated with both LT-IIb-B5 and LT-IIb that are statistically significantly different (*, p < 0.05) from those treated with LT-IIb-B5 alone are indicated. Statistically significant (•, p < 0.05) reversal of the LT-IIb effects by SQ22536 (A and B) or H8910 (C and D) is indicated.

 
The same groups of cells were also analyzed for IL-10 production, and SQ22536 was similarly found to significantly reverse (p < 0.05) the ability of LT-IIb to up-regulate LT-IIb-B5-induced IL-10 (Fig. 2B). LT-IIb induced IL-10 on its own, although production was diminished in the presence of SQ22536 (Fig. 2B). The apparent involvement of cAMP in these regulatory effects, and the fact that PKA is a predominant effector of cAMP signaling (25), prompted us to investigate whether a PKA inhibitor, H89 (24), could replicate our observations with SQ22536. Indeed, we found that H89 significantly (p < 0.05) reversed the abilities of LT-IIb to inhibit LT-IIb-B5-induced TNF-{alpha} production (Fig. 2C) and to up-regulate LT-IIb-B5-induced IL-10 production (Fig. 2D). The Fig. 2 data collectively suggest that cAMP-dependent PKA mediates the regulatory effects of LT-IIb on cytokine induction by LT-IIb-B5.

LT-IIb does not stimulate TLR2/1 recruitment or activation but inhibits TLR2/1-mediated NF-{kappa}B activation by LT-IIb-B5

We next investigated whether the ability of LT-IIb-B5 to induce TLR2/1-mediated activation of NF-{kappa}B (7) is inhibited by LT-IIb in a cAMP- and PKA-dependent way. For this purpose, we used HEK-293 cells cotransfected with human TLR2 and its signaling partner TLR1 (required for cooperative signaling in response to LT-IIb-B5 (8)) and with a NF-{kappa}B reporter system. We then determined the ability of LT-IIb-B5 to activate NF-{kappa}B-dependent transcription of a luciferase gene in the presence or absence of LT-IIb, with or without pretreatment with SQ22536 or H89. In contrast to LT-IIb, LT-IIb-B5 readily induced NF-{kappa}B-dependent transcription in TLR2/1-transfected cells (but not in empty vector-transfected controls; data not shown). However, the TLR2/1 stimulatory activity of LT-IIb-B5 was significantly inhibited (p < 0.05) by LT-IIb (Fig. 3). Pretreatment with SQ22536 or H89 significantly reversed the inhibitory effect of LT-IIb on NF-{kappa}B activation (Fig. 3) thus suggesting that cAMP-dependent PKA signaling by LT-IIb antagonizes TLR2-mediated activation of NF-{kappa}B. Boiling of LT-IIb-B5 or LT-IIb resulted in loss of their activities, in that boiled LT-IIb-B5 did not induce TLR2/1-mediated NF-{kappa}B-activation, whereas boiled LT-IIb did not inhibit TLR2/1-mediated NF-{kappa}B-activation by unboiled LT-IIb-B5 (data not shown).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 3. LT-IIb antagonizes TLR2/1-dependent NF-{kappa}B activation by LT-IIb-B5 through a cAMP/PKA mechanism. HEK-293 cells cotransfected with TLR2/TLR1 and NF-{kappa}B reporter system were exposed to 2 µg/ml LT-IIb-B5, LT-IIb, or a combination of the two, with or without 30-min pretreatment with SQ22536 (200 µM) or with H89 (10 µM). Following a 6-h stimulation period, NF-{kappa}B-dependent transcription of a luciferase reporter gene was determined as relative luciferase activity (RLA) normalized to that of unstimulated cells. Results are presented as mean ± SD (n = 3) from one of two experiments that yielded similar findings. Statistically significant (*, p < 0.05) inhibition of LT-IIb-B5-induced cell activation in the presence of LT-IIb and significant (•, p < 0.05) reversal of the LT-IIb effect are indicated.

 
Another possible mechanism accounting for the contrasting abilities of LT-IIb and LT-IIb-B5 in TLR2/1-mediated activation of NF-{kappa}B may involve differential recruitment of TLR2 and TLR1 to lipid rafts, which serve as membrane platforms for TLR-mediated signaling (22, 26, 27). This mechanism was examined using human monocytes and FRET. Specifically, we investigated possible TLR2 and TLR1 associations with ganglioside GM1, an established lipid raft marker. Indeed, GM1 is found constitutively and in abundance in lipid rafts and the use of FRET has demonstrated that GM1 associates with recruitable receptors in correlation with biochemical data on lipid raft composition (22). We observed significant (p < 0.05) energy transfer between TLR2 and GM1, as well as between TLR1 and GM1, in LT-IIb-B5-stimulated cells compared with LT-IIb-treated or unstimulated cells (Fig. 4A). In contrast, FRET values for possible GM1 associations with TLR4 or MHC class I (negative controls) were consistently low regardless of the cellular activation state (Fig. 4A). These data indicate that LT-IIb-B5 induces TLR2/1-GM1 associations and, therefore, TLR2 and TLR1 (but not TLR4 or MHC class I) are mobilized to lipid rafts in response to LT-IIb-B5. The involvement of lipid rafts in LT-IIb-B5-induced TLR2/1-GM1 coassociations was further supported by the finding that cell pretreatment with MCD, which disrupts lipid raft organization, abrogated energy transfer between GM1 and TLR2 or TLR1 (Fig. 4A).


Figure 4
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 4. Differential abilities of LT-IIb and LT-IIb-B5 for inducing TLR2/1-ganglioside associations. Human monocytes were untreated or pretreated for 30 min with 10 mM MCD and then stimulated with LT-IIb or LT-IIb-B5. FRET was used to measure energy transfer between the indicated receptors and GM1 (A) or GD1a (B). Results are shown as the mean ± SD of the percentage of energy transfer, calculated from three independent experiments. The maximum and minimum energy transfer efficiencies in the system were 37 ± 1.5 and 4 ± 1, respectively (data not shown), and were determined as the energy transfer between two different epitopes on the same molecule (CD14) (maximum) or between molecules that do not engage in heterotypic associations (CD14 and MHC class I) (minimum). Significantly higher activities (*, p < 0.05) by LT-IIb-B5 compared with LT-IIb are indicated. Significant reversal (•, p < 0.05) of energy transfer increase due to MCD pretreatment is shown.

 
We next compared the LT-IIb-B5 and LT-IIb for their abilities to induce association of TLR2 and TLR1 with ganglioside GD1a, which plays an important coreceptor function for TLR2 activation by LT-IIb-B5 (28). We reasoned that possible inability of LT-IIb to induce TLR2/1-GD1a association would constitute another potential mechanism for its failure to induce significant TLR2/1-dependent NF-{kappa}B activation (Fig. 3). Indeed, we observed significant differences (p < 0.05) between LT-IIb-B5 and LT-IIb regarding their abilities to induce energy transfer between GD1a and TLR2 or GD1a and TLR1, indicating that the B pentamer is more potent in inducing TLR2/1-GD1a associations (Fig. 4B). However, cell pretreatment with MCD abrogated energy transfer between GD1a and TLR2 or GD1a and TLR1 (Fig. 4B). As expected, none of the molecules induced energy transfer between GD1a and TLR4, which was used as a negative control (Fig. 4B).

The relative inability of LT-IIb to recruit TLR2 was consistent with additional observations that the capacity of this holotoxin to induce certain cytokines is TLR2-independent. Specifically, whereas the ability of LT-IIb-B5 to induce IL-10 was abrogated in macrophages deficient in TLR2 or in both TLR2 and TLR4 (but not in TLR4-deficient or wild-type macrophages), IL-10 induction by the LT-IIb holotoxin was not affected by any of these TLR deficiencies (Fig. 5, A and B). We have also detected low but measurable IL-6 levels induced by LT-IIb that were similarly independent of TLR2 activation, in stark contrast to IL-6 induction by LT-IIb-B5 (Fig. 5, C and D).


Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 5. Differential requirement for TLR2 in cytokine induction by LT-IIb and LT-IIb-B5. Mouse macrophages from wild-type (WT) mice or mice deficient in TLR2, TLR4, or both TLR2 and TLR4 (TLR2/4) were stimulated for 16 h with LT-IIb and LT-IIb-B5 (2 µg/ml). Induction of IL-10 (A and B) or IL-6 (C and D) release in culture supernatants was assayed by ELISA. Results are presented as mean ± SD (n = 3) from one of two independent sets of experiments yielding similar findings. Statistically significant (*, p < 0.05) inhibition of cytokine release in TLR-deficient cells compared with corresponding wild-type controls is indicated.

 
Construction of mutagenically detoxified mutants of LT-IIb

We next constructed catalytic mutants of LT-IIb for functional comparison with the wild-type molecule. The rationale was to provide definitive evidence implicating the catalytic activity LT-IIb in inhibition of cell activation (by LT-IIb-B5 or other stimuli). Moreover, such constructs would allow us to determine whether abrogation of catalytic activity transforms the mutant LT-IIb holotoxins into proinflammatory molecules. Because the Ser residue at position 59 of type II enterotoxins (equivalent to Ser residue a position 61 of type I enterotoxins) is considered critical for catalytic activity (1), we have engineered a single point substitution mutation (S59K) in the catalytic domain of the A subunit of LT-IIb. Although this mutant displayed diminished toxicity in the Y1 adrenal biotoxicity assay (1), it retained cAMP-inducing activity in RAW264.7 mouse macrophages (data not shown). We therefore constructed a dual point substitution mutation (S59K/E110K) (Glu110 being equivalent to the catalytic residue Glu112 of type I enterotoxins (2)) and the resulting mutant (1 µg/ml) displayed diminished cAMP-elevating capacity compared with the single point mutant or wild-type LT-IIb, which induced cAMP at levels similar to those induced by 20 µM forskolin (Fig. 6A). The S59K/E110K mutant displayed negligible capacity to elevate cAMP over basal levels (Fig. 6A); this result is unlikely to reflect residual catalytic activity because LT-IIb-B5 behaved similarly to LT-IIb(S59K/E110K). In this regard, we have shown that low-level cAMP induction by LT-IIb-B5 probably involves TLR2-dependent production of PGE2 (Fig. 6B, inset) because inhibition of PGE2 synthesis by indomethacin also inhibited cAMP induction by LT-IIb-B5 (Fig. 6B).


Figure 6
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 6. Induction of cAMP production by wild-type LT-IIb, catalytic mutants, and LT-IIb-B5. A, RAW264.7 mouse macrophages were stimulated with forskolin (20 µM), wild-type LT-IIb, single or dual point catalytic mutants (S59K and S59K/E110K), or LT-IIb-B5 (all at 1 µg/ml) and assayed for induction of intracellular cAMP levels using a cAMP enzyme immunoassay kit (Cayman Chemicals). B, LT-IIb-B5-stimulated macrophages were assayed for intracellular cAMP as above, and also for induction of PGE2 in culture supernatants by ELISA. Before LT-IIb-B5 stimulation or treatment with medium only (M), the cells were pretreated with indomethacin (IND, 1 µM) to determine whether inhibition of PGE2 synthesis affects cAMP induction. TLR2-dependence of LT-IIb-B5-induced PGE2 (inset) using wild-type and TLR2-deficient macrophages was determined. Results are presented as mean ± SD (n = 3) from typical experiments. Statistically significant decrease of cAMP induction compared with wild-type LT-IIb (*, p < 0.05) or compared with the single point mutant (S59K) (•, p < 0.05) is shown. Significant induction of cAMP or PGE2 (*, p < 0.05) compared with medium-treated cells and significant reversal of these activities by indomethacin (•, p < 0.05) are shown.

 
Comparison of LT-IIb with catalytic mutants for inhibitory activity in TNF-{alpha} induction

We reasoned that if cAMP is critical for mediating the inhibitory effect of LT-IIb on TNF-{alpha} induction by LT-IIb-B5 (as suggested by the data in Fig. 2), then the S59K/E110K mutant should lose this inhibitory activity. If in contrast, the mutant retained partial inhibitory activity, this would suggest an additional, cAMP-independent effect on suppressing TNF-{alpha} induction. In this context, we performed a functional comparison between wild-type LT-IIb and both catalytically defective mutants (S59K and S59K/E110K). As expected, wild-type LT-IIb inhibited TNF-{alpha} induction in J774A.1 mouse macrophages stimulated by LT-IIb-B5 (Fig. 7A) or E. coli LPS (Fig. 7B). In striking contrast, LT-IIb(S59K) and LT-IIb(S59K/E110K) were without any effect in that regard (Fig. 7, A and B). The inability of LT-IIb(S59K/E110K) to inhibit TNF-{alpha} induction is consistent with the notion that the catalytic activity of the holotoxin is critical for this inhibitory effect. Apparently, the residual catalytic activity of the single point mutant (S59K) (Fig. 7A) was insufficient to inhibit, even partially, TNF-{alpha} induction by LT-IIb-B5 or LPS (Fig. 7, A and B), suggesting that relatively high cAMP levels may be required for this inhibitory activity. Additional evidence that cAMP elevation may underlie the inhibitory effects of LT-IIb on LT-IIb-B5- and LPS-induced TNF-{alpha} production was obtained in experiments using forskolin, which causes elevation of intracellular cAMP by directly activating adenylate cyclase, or using dibutyryl cAMP, which acts as a membrane-permeable cAMP analog (29). Both forskolin and dibutyryl cAMP could mimic the LT-IIb inhibitory effects on TNF-{alpha} induction by LT-IIb-B5 (Fig. 8A) or LPS (Fig. 8B) in a dose-dependent way.


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 7. Inhibition of LT-IIb-B5-induced or E. coli LPS-induced TNF-{alpha} by wild-type but not by catalytically defective mutants of LT-IIb. J774A.1 mouse macrophages were maintained unstimulated or stimulated with 2 µg/ml LT-IIb-B5 (A) or 100 ng/ml E. coli LPS (B) in the absence or presence of 2 µg/ml wild-type LT-IIb or catalytically defective mutants (S59K or S59K/E110K). C, Macrophages were incubated with the indicated concentrations of wild-type or mutant LT-IIb. After 16-h incubation, induction of TNF-{alpha} release in culture supernatants was assayed by ELISA. Results are presented as mean ± SD (n = 3) from one of three independent sets of experiments yielding similar results. Statistically significant inhibition of LT-IIb-B5-induced (*, p < 0.05) or LPS-induced TNF-{alpha} production is shown. Significantly higher TNF-{alpha} induction (•, p < 0.05) by the mutants compared with wild-type LT-IIb and between the mutants ({triangledown}, p < 0.05) are denoted.

 

Figure 8
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 8. cAMP-mediated inhibition of LT-IIb-B5-induced or E. coli LPS-induced TNF-{alpha}. J774A.1 mouse macrophages were stimulated with LT-IIb-B5 (2 µg/ml) (A) or E. coli LPS (10 ng/ml) (B) with or without 30-min pretreatment with the indicated concentrations of forskolin or dibutyryl (dbt) cAMP. Induction of TNF-{alpha} release in culture supernatants was assayed by ELISA, and values were normalized to those of uninhibited controls, the value of which was taken as 100. Results are presented as mean ± SD (n = 3) from one of three independent sets of experiments yielding similar findings. Statistically significant (*, p < 0.05) inhibition of TNF-{alpha} induction is indicated.

 
Enhanced TLR2-binding activity by LT-IIb-B5 compared with wild-type or mutant LT-IIb holotoxins

In the previous experiment (Fig. 7), a graded effect was observed in the abilities of wild-type and mutant LT-IIb molecules for cytokine induction on their own, increasing in inverse relationship to their cAMP-inducing activities. Specifically, the dual point mutant induced significantly higher (p < 0.05) TNF-{alpha} levels compared with the other two molecules, whereas the single point mutant was significantly more proinflammatory (p < 0.05) than wild-type LT-IIb, which induced little or no TNF-{alpha} (Fig. 7, A and B). These effects were confirmed in an independent dose-response experiment, although none of the mutants approached TNF-{alpha} induction by LT-IIb-B5 (Fig. 7C). Therefore, although the catalytic activity of LT-IIb is essential for inhibiting TNF-{alpha} induction by other stimuli, abrogation of this catalytic activity in LT-IIb(S59K/E110K) has not rendered the mutant as effective as LT-IIb-B5 in TNF-{alpha} induction. This implies a catalytic-independent, yet still A subunit-dependent mechanism that prevents the S59K/E110K mutant from inducing cellular activation with comparable potency to LT-IIb-B5. In this regard, we examined the TLR2-binding activity of LT-IIb-B5 in comparison to the mutants as well as the wild-type LT-IIb. The rationale for this experiment involved the possibility that the A subunit may sterically interfere with TLR2 binding. We found that LT-IIb-B5 displayed significantly increased (p < 0.05) binding activity for TLR2 compared with LT-IIb or the catalytic mutants, which displayed minimal binding (Fig. 9A). All molecules displayed similar binding to GD1a (positive control) but their binding to CD14 (negative control) was negligible or undetectable (Fig. 9A). In a competitive inhibition assay, the TLR2-binding activity of biotinylated LT-IIb-B5 was antagonized by unlabeled LT-IIb-B5 in a dose-dependent way (p < 0.05) (Fig. 9B), further confirming binding specificity. We next examined the binding of LT-IIb-B5 to TLR2 using a cell-based approach. Specifically, LT-IIb-B5 displayed significantly higher binding (p < 0.05) to TLR2-transfected CHO-K1 cells than to empty vector-transfected cells (Fig. 9C), in contrast to LT-IIb, which did not show significant binding to TLR2-transfected relative to empty vector-transfected cells (data not shown). The specificity of LT-IIb-B5 binding to TLR2 was verified by the findings that anti-TLR2 mAb (but not IgG2a isotype control) significantly inhibited (p < 0.05) binding and, moreover, LT-IIb-B5 failed to bind TLR4-transfected CHO-K1 cells (Fig. 9C). Furthermore, the binding of LT-IIb-B5 (used in biotinylated form for probing with streptavidin-FITC) was diminished by 100-fold excess unlabeled (nonbiotinylated) LT-IIb-B5, whereas it was unaffected in the presence of similar excess of unlabeled LT-IIb (Fig. 9C).


Figure 9
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 9. Differential abilities of wild-type or mutant LT-IIb and LT-IIb-B5 for TLR2 binding. A, Binding of LT-IIb or LT-IIb-B5 (10 µg/ml) to the indicated receptors was determined on receptor-coated microtiter wells probed with anti-LT-IIb Ab. Following addition of peroxidase-conjugated secondary Ab, binding was measured colorimetrically. B, Binding of biotinylated LT-IIb-B5 (10 µg/ml) to plate-bound TLR2 in the presence of excess unlabeled LT-IIb-B5 at the indicated concentrations. Bound protein was probed with peroxidase-conjugated streptavidin. C, CHO-K1 cells were transfected with empty vector or plasmids encoding human TLR2 or TLR4 (coexpressed with MD2). The cells were incubated with biotinylated LT-IIb-B5 (1 µg/ml), in the presence or absence of anti-TLR2 mAb or IgG2a isotype control (10 µg/ml), or 100-fold excess of unlabeled LT-IIb-B5 or LT-IIb, and binding was measured as cell-associated fluorescence in relative fluorescence units (RFU) after staining with FITC-conjugated streptavidin. Data are shown as mean ± SD of triplicate determinations from typical experiments with similar findings. Significantly higher LT-IIb-B5 binding (*, p < 0.05) compared with wild-type or mutant LT-IIb is shown in A. Significant (*, p < 0.05) inhibition of binding is indicated in B. In C, significant binding compared with vector-transfected cells (*, p < 0.05), and significant reversal of binding (•, p < 0.05) are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The findings of this study suggests that the A subunit of LT-IIb is associated with reduced cell activation through both cAMP-dependent and cAMP-independent inhibitory effects. The investigation was facilitated by comparing catalytically defective mutants with wild-type LT-IIb or with its B pentamer LT-IIb-B5. The comparative data indicate that the catalytic activity of LT-IIb is essential for its antagonistic effect on LT-IIb-B5-induced cell activation, as determined by NF-{kappa}B-dependent transcription and TNF-{alpha} induction. However, the catalytic activity of LT-IIb is not totally responsible for the self-inhibitory effect of LT-IIb on TNF-{alpha} induction because, otherwise, LT-IIb(S59K/E110K) would display a similar TNF-{alpha}-inducing activity to that of LT-IIb-B5. In an analogous context, inhibition of cAMP synthesis by SQ22536 completely reversed the inhibitory effect of LT-IIb on LT-IIb-B5-induced TNF-{alpha}, yet it had a weak effect on augmenting the ability of LT-IIb to induce TNF-{alpha} (Fig. 2A).

The observations we discussed suggest that the A subunit can mediate inhibitory effects in a catalytic-independent way, although the precise mechanism is currently uncertain. Some insights, however, may be gleaned from the crystallographic structure of LT-IIb (2). LT-IIb-B5 and other B pentamers of heat-labile enterotoxins interact via both hydrophobic and hydrophilic interactions with other molecules (2, 30). The so-called "lower end" of the B pentamer pore of LT-IIb interacts with the oligosaccharide moiety of GD1a through hydrophilic interactions, whereas the "upper end" of the pore contains a large hydrophobic surface (549-Å2), which interacts with the A2 segment of the A subunit. Because this hydrophobic surface area is solvent accessible in the absence of the A subunit (2, 30), it is possible that the isolated B pentamer may interact hydrophobically with molecules such as TLRs that also depend on hydrophobic interactions for ligand binding (31, 32, 33). Presumably, the presence of the A subunit in the holotoxin could preclude or suppress TLR interactions. Our findings that LT-IIb-B5 interacts more readily than the LT-IIb holotoxin or its catalytic mutants with TLR2 (Fig. 9) are consistent with the discussed considerations. Interestingly, the notion that the A subunit is responsible for the differential potential between the holotoxin and its B pentamer for TLR2 interactions (Fig. 1) is analogous to another such example involving a nonenterotoxin TLR2 agonist, mycobacterial lipoarabinomannan. Specifically, it has been proposed that steric hindrance by the arabinan moiety blocks the TLR2 agonistic activity of other lipoarabinomannan components that are potentially accessible for TLR2 recognition, thereby leading to reduced proinflammatory cytokine production (34). This activity may represent a mycobacterial strategy for evading immune surveillance given that pathogenic mycobacteria are typically anti-inflammatory, whereas nonpathogenic strains are proinflammatory (34).

Our observations that the LT-IIb holotoxin suppresses its own proinflammatory potential, and antagonizes the proinflammatory activities of LT-IIb-B5 and LPS, could similarly suggest a potential immune evasion mechanism by enterotoxic E. coli. Our in vitro model (Fig. 1) predicts that LT-IIb inhibits TLR-dependent NF-{kappa}B activation and induction of proinflammatory cytokines via a cAMP-dependent mechanism. This experiment is consistent with published reports on the in vivo anti-inflammatory properties of a related cAMP-inducing AB5 enterotoxin. Specifically, cholera toxin is noninflammatory in an animal model at doses that readily induce intestinal fluid secretion (35). Moreover, in humans, V. cholerae induces either noninflammatory diarrhea or inflammatory gastroenteritis, depending upon the presence of a single virulence factor, cholera toxin; indeed, cholera toxin-producing strains induce watery diarrhea in the absence of inflammation, whereas strains that do not produce cholera toxin cause inflammatory diarrhea (36).

The relative inability of the LT-IIb holotoxin to bind TLR2 may be linked to its inefficiency in inducing lipid raft recruitment of TLR2, which like other surface TLRs can potentially be mobilized to the rafts upon activation with appropriate ligands (22, 37). We believe that at least a mechanism whereby a given ligand can recruit and retain a TLR to lipid rafts involves the ability of the ligand to bind both a lipid raft molecule (GD1a, in the case of LT-IIb) and the TLR in question. Unlike LT-IIb-B5, which binds both GD1a and TLR2, LT-IIb can efficiently bind only GD1a and thus cannot readily induce formation of a TLR2-GD1a complex in lipid rafts.

Therefore, the signal transduction pathway activated by the LT-IIb holotoxin should essentially be TLR2-independent and primarily dependent upon its ability to induce elevation of intracellular cAMP (Fig. 1). Conclusive evidence that LT-IIb is quite poor in inducing TLR2 signaling, if at all, was obtained by examining its ability to induce IL-10. Importantly, the induction of this cytokine is not inhibited by cAMP and thus monitoring possible TLR2-dependent induction of IL-10 by LT-IIb cannot be masked by cAMP interference. Although both LT-IIb and LT-IIb-B5 induced comparable IL-10 levels, their mechanisms were clearly distinct involving TLR2-independent and TLR2-dependent signaling, respectively (Fig. 5, A and B). In fact, the involvement of distinct mechanisms may explain the synergism between LT-IIb and LT-IIb-B5 in IL-10 induction (Fig. 2, B and D). This synergism, as well as the ability of LT-IIb to induce IL-10 on its own, is likely dependent on the cAMP/PKA pathway because the activities are abrogated by SQ22536 or H89 (Fig. 2, B and D). Similarly, the ability of HIV-1 gp120-gp41 complex to induce IL-10 is attributed to adenylate cyclase-mediated production of cAMP and PKA activation (38, 39). Mechanistically, cAMP-dependent PKA can enhance IL-10 transcription through CREB activation (40, 41). Although LT-IIb inhibits NF-{kappa}B activation and TNF-{alpha} induction through a predominantly IL-10-independent manner (7), the inhibitory mechanism is abrogated by SQ22636 or H89 (Figs. 1 and 2) and thus may also involve the cAMP-PKA pathway (Fig. 1). In this regard, cAMP-dependent PKA is known to phosphorylate CREB, which can then effectively compete with the p65 subunit of NF-{kappa}B for limiting amounts of a common transcriptional coactivator, the CREB-binding protein (42). Consequently, this leads to decreased NF-{kappa}B activation and reduced transcription of NF-{kappa}B-dependent cytokine genes like TNF-{alpha}.

The immunomodulatory differences between LT-IIb and LT-IIb-B5 may have important implications also for their adjuvant properties. LT-IIb-B5 may conceivably enhance adaptive immunity through TLR2 stimulation; in this regard, TLR2 and other TLRs function as adjuvant receptors through induction of costimulatory molecules and immunoenhancing cytokines by APC (43, 44). In contrast, the LT-IIb holotoxin may exert adjuvant action in a noninflammatory manner mediated by its ganglioside-binding and toxic-catalytic activities, although type I and type II holotoxins arguably contain A subunit-dependent adjuvanticity that is independent of catalytic activity (reviewed in Ref. 6). In stark contrast to TLR adjuvants, the type I and type II enterotoxins (e.g., cholera toxin, LT-I, LT-IIa, and LT-IIb) can elicit adjuvant activity in the absence of proinflammatory activity (6, 36, 45, 46, 47). Cholera toxin, a prototypical cAMP-inducing enterotoxin that has been studied in great molecular detail, promotes the induction of type 1 regulatory T cells as well as Th2 cells, whereas it proactively inhibits Th1 cell differentiation and induction of proinflammatory cytokines, such as IL-12 and TNF-{alpha} (36, 45, 47, 48). Despite lack of proinflammatory activity, cholera toxin and E. coli enterotoxins induce enhanced Ag presentation by a variety of APC types and promote Ig isotype differentiation in B cells leading to increased mucosal IgA production (6, 45, 48). Thus, induction of proinflammatory signaling is not an obligatory mechanism for adjuvanticity, and in this regard the LT-IIb and its proinflammatory derivative, LT-IIb-B5, appear to exert distinct immunomodulatory effects. Using an intranasal mouse immunization model, we have compared LT-IIb and LT-IIb-B5 as mucosal adjuvants for a coadministered protein Ag. Both molecules potentiated specific mucosal Ab responses to the coadministered immunogen (our unpublished observations) and we will further determine whether the adjuvant effects of LT-IIb and LT-IIb-B5 are TLR2-independent and TLR2-dependent, respectively.

In summary, our results underscore the importance of the A subunit of LT-IIb in determining the state of cell activation through both cAMP-dependent and –independent effects (Fig. 1). Whereas the A subunit mediates the ability of the holotoxin to elevate intracellular cAMP, activate cAMP-dependent PKA, and inhibit NF-{kappa}B-dependent transcription, the absence of the A subunit from LT-IIb-B5 allows more efficient interaction with TLR2/1 and induction of downstream signaling for NF-{kappa}B activation (Fig. 1). These findings provide mechanistic insights for better understanding enterotoxic E. coli pathogenesis as well as for exploiting LT-IIb and derivatives for adjuvant development.


   Acknowledgment
 
We thank Dr. Sergio Arce (State University of New York, Buffalo, NY) for critical review of the manuscript.


   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 U.S. Public Health Service Grants DE06746 (to M.W.R.), DE13833 (to T.D.C.), and DE015254 and DE017138 (to G.H.) from the National Institutes of Health, and by the Wellcome Trust and the Heart Research Fund (to K.T.). Back

2 Address correspondence and reprint requests to Dr. George Hajishengallis, Department of Microbiology and Immunology, University of Louisville Health Sciences Center, 501 South Preston Street, Room 206, Louisville, KY 40292. E-mail address: g0haji01{at}louisville.edu Back

3 Abbreviations used in this paper: PKA, protein kinase A; MCD, methyl-beta-cyclodextrin; HEK, human embryonic kidney; CHO, Chinese hamster ovary; FRET, fluorescence resonance energy transfer. Back

Received for publication December 8, 2006. Accepted for publication January 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Holmes, R., M. G. Jobling, T. Connell. 1995. Cholera toxin and related enterotoxins of Gram-negative bacteria. J. Moss, and B. Iglewski, and M. Vaughn, and A. T. Tu, eds. Handbook of Natural Toxins: Bacterial Toxins and Virulence Factors in Disease 225-255. Marcel Dekker, New York.
  2. Van Den Akker, F., S. Sarfaty, E. M. Twiddy, T. Connell, R. K. Holmes, W. G. J. Hol. 1996. Crystal structure of a new heat-labile enterotoxin, LTIIb. Structure 4: 665-678. [Medline]
  3. Gill, D. M., J. D. Clements, D. C. Robertson, R. A. Finkelstein. 1981. Subunit number and arrangement in Escherichia coli heat-labile enterotoxin. Infect. Immun. 33: 677-682. [Abstract/Free Full Text]
  4. Sonnino, S., D. Acquotti, L. Riboni, A. Giuliani, G. Kirschner, G. Tettamanti. 1986. New chemical trends in ganglioside research. Chem. Phys. Lipids 42: 3-26. [Medline]
  5. Lalli, E., P. Sassone-Corsi. 1994. Signal transduction and gene regulation: the nuclear response to cAMP. J. Biol. Chem. 269: 17359-17362. [Free Full Text]
  6. Hajishengallis, G., S. Arce, C. M. Gockel, T. D. Connell, M. W. Russell. 2005. Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections. J. Dent. Res. 84: 1104-1116. [Abstract/Free Full Text]
  7. Hajishengallis, G., H. Nawar, R. I. Tapping, M. W. Russell, T. D. Connell. 2004. The Type II heat-labile enterotoxins LT-IIa and LT-IIb and their respective B pentamers differentially induce and regulate cytokine production in human monocytic cells. Infect. Immun. 72: 6351-6358. [Abstract/Free Full Text]
  8. Hajishengallis, G., R. I. Tapping, M. H. Martin, H. Nawar, E. A. Lyle, M. W. Russell, T. D. Connell. 2005. Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins. Infect. Immun. 73: 1343-1349. [Abstract/Free Full Text]
  9. Kopp, E., R. Medzhitov. 2003. Recognition of microbial infection by Toll-like receptors. Curr. Opin. Immunol. 15: 396-401. [Medline]
  10. Sansonetti, P.. 2002. Host-pathogen interactions: the seduction of molecular cross talk. Gut 50: (Suppl 3):III2-III8. [Medline]
  11. Fukuta, S., J. L. Magnani, E. M. Twiddy, R. K. Holmes, V. Ginsburg. 1988. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LTIIa, and LTIIb. Infect. Immun. 56: 1748-1753. [Abstract/Free Full Text]
  12. Nawar, H. F., S. Arce, M. W. Russell, T. D. Connell. 2005. Mucosal adjuvant properties of mutant LT-IIa and LT-IIb enterotoxins that exhibit altered ganglioside-binding activities. Infect. Immun. 73: 1330-1342. [Abstract/Free Full Text]
  13. Harokopakis, E., M. H. Albzreh, M. H. Martin, G. Hajishengallis. 2006. TLR2 transmodulates monocyte adhesion and transmigration via Rac1- and PI3K-mediated inside-out signaling in response to Porphyromonas gingivalis fimbriae. J. Immunol. 176: 7645-7656. [Abstract/Free Full Text]
  14. Wooten, R. M., Y. Ma, R. A. Yoder, J. P. Brown, J. H. Weis, J. F. Zachary, C. J. Kirschning, J. J. Weis. 2002. Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi. J. Immunol. 168: 348-355. [Abstract/Free Full Text]
  15. Vabulas, R. M., P. Ahmad-Nejad, S. Ghose, C. J. Kirschning, R. D. Issels, H. Wagner. 2002. HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277: 15107-15112. [Abstract/Free Full Text]
  16. Hajishengallis, G., P. Ratti, E. Harokopakis. 2005. Peptide mapping of bacterial fimbrial epitopes interacting with pattern recognition receptors. J. Biol. Chem. 280: 38902-38913. [Abstract/Free Full Text]
  17. Hajishengallis, G., R. I. Tapping, E. Harokopakis, S.-I. Nishiyama, P. Ratti, R. E. Schifferle, E. A. Lyle, M. Triantafilou, K. Triantafilou, F. Yoshimura. 2006. Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus. Cell. Microbiol. 8: 1557-1570. [Medline]
  18. Harokopakis, E., G. Hajishengallis. 2005. Integrin activation by bacterial fimbriae through a pathway involving CD14, Toll-like receptor 2, and phosphatidylinositol-3-kinase. Eur. J. Immunol. 35: 1201-1210. [Medline]
  19. Massari, P., A. Visintin, J. Gunawardana, K. A. Halmen, C. A. King, D. T. Golenbock, L. M. Wetzler. 2006. Meningococcal porin PorB binds to TLR2 and requires TLR1 for signaling. J. Immunol. 176: 2373-2380. [Abstract/Free Full Text]
  20. Hajishengallis, G., M. Wang, E. Harokopakis, M. Triantafilou, K. Triantafilou. 2006. Porphyromonas gingivalis fimbriae proactively modulate beta2 integrin adhesive activity and promote binding to and internalization by macrophages. Infect. Immun. 74: 5658-5666. [Abstract/Free Full Text]
  21. Triantafilou, K., M. Triantafilou, R. L. Dedrick. 2001. A CD14-independent LPS receptor cluster. Nat. Immunol. 2: 338-345. [Medline]
  22. Triantafilou, M., K. Miyake, D. T. Golenbock, K. Triantafilou. 2002. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation. J. Cell Sci. 115: 2603-2611. [Abstract/Free Full Text]
  23. Triantafilou, M., K. Brandenburg, S. Kusumoto, K. Fukase, A. Mackie, U. Seydel, K. Triantafilou. 2004. Combinational clustering of receptors following stimulation by bacterial products determines lipopolysaccharide responses. Biochem. J. 381: 527-536. [Medline]
  24. Armstrong, R. A.. 1995. Investigation of the inhibitory effects of PGE2 and selective EP agonists on chemotaxis of human neutrophils. Br. J. Pharmacol. 116: 2903-2908. [Medline]
  25. Smith, F. D., J. D. Scott. 2006. Anchored cAMP signaling: onward and upward. A short history of compartmentalized cAMP signal transduction. Eur. J. Cell Biol. 85: 585-592. [Medline]
  26. Soong, G., B. Reddy, S. Sokol, R. Adamo, A. Prince. 2004. TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells. J. Clin. Invest. 113: 1482-1489. [Medline]
  27. Cherukuri, A., M. Dykstra, S. K. Pierce. 2001. Floating the raft hypothesis: lipid rafts play a role in immune cell activation. Immunity 14: 657-660. [Medline]
  28. Liang, S., M. Wang, R. I. Tapping, V. Stepensky, H. F. Nawar, M. Triantafilou, K. Triantafilou, T. D. Connell, and G. Hajishengallis. 2007. Ganglioside GD1a is an essential coreceptor for Toll-like receptor 2 signaling in response to the B subunit of type IIB enterotoxin. J. Biol. Chem. 282: In press.
  29. Suzuki, S., I. Yamamoto, J. Arita. 1999. Mitogen-activated protein kinase-dependent stimulation of proliferation of rat lactotrophs in culture by 3',5'-cyclic adenosine monophosphate. Endocrinology 140: 2850-2858. [Abstract/Free Full Text]
  30. Tinker, J. K., J. L. Erbe, W. G. Hol, R. K. Holmes. 2003. Cholera holotoxin assembly requires a hydrophobic domain at the A-B5 interface: mutational analysis and development of an in vitro assembly system. Infect. Immun. 71: 4093-4101. [Abstract/Free Full Text]
  31. Okusawa, T., M. Fujita, J. Nakamura, T. Into, M. Yasuda, A. Yoshimura, Y. Hara, A. Hasebe, D. T. Golenbock, M. Morita, et al 2004. Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by Toll-like receptors 2 and 6. Infect. Immun. 72: 1657-1665. [Abstract/Free Full Text]
  32. Nishiguchi, M., M. Matsumoto, T. Takao, M. Hoshino, Y. Shimonishi, S. Tsuji, N. A. Begum, O. Takeuchi, S. Akira, K. Toyoshima, T. Seya. 2001. Mycoplasma fermentans lipoprotein M161Ag-induced cell activation is mediated by Toll-like receptor 2: role of N-terminal hydrophobic portion in its multiple functions. J. Immunol. 166: 2610-2616. [Abstract/Free Full Text]
  33. Seong, S. Y., P. Matzinger. 2004. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat. Rev. Immunol. 4: 469-478. [Medline]
  34. Quesniaux, V. J., D. M. Nicolle, D. Torres, L. Kremer, Y. Guerardel, J. Nigou, G. Puzo, F. Erard, B. Ryffel. 2004. Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans. J. Immunol. 172: 4425-4434. [Abstract/Free Full Text]
  35. Triadafilopoulos, G., C. Pothoulakis, R. Weiss, C. Giampaolo, J. T. Lamont. 1989. Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum. Gastroenterology 97: 1186-1192. [Medline]
  36. Fullner Satchell, K. J.. 2003. Activation and suppression of the proinflammatory immune response by Vibrio cholerae toxins. Microb. Infect. 5: 1241-1247. [Medline]
  37. Pfeiffer, A., A. Bottcher, E. Orso, M. Kapinsky, P. Nagy, A. Bodnar, I. Spreitzer, G. Liebisch, W. Drobnik, K. Gempel, et al 2001. Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts. Eur. J. Immunol. 31: 3153-3164. [Medline]
  38. Barcova, M., C. Speth, L. Kacani, F. Uberall, H. Stoiber, M. P. Dierich. 1999. Involvement of adenylate cyclase and p70S6-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1. Eur. J. Physiol. 437: 538-546. [Medline]
  39. Masci, A. M., M. Galgani, S. Cassano, S. De Simone, A. Gallo, V. De Rosa, S. Zappacosta, L. Racioppi. 2003. HIV-1 gp120 induces anergy in naive T lymphocytes through CD4-independent protein kinase-A-mediated signaling. J. Leukocyte Biol. 74: 1117-1124. [Abstract/Free Full Text]
  40. Platzer, C., E. Fritsch, T. Elsner, M. H. Lehmann, H. D. Volk, S. Prosch. 1999. Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells. Eur. J. Immunol. 29: 3098-3104. [Medline]
  41. Hu, X., P. K. Paik, J. Chen, A. Yarilina, L. Kockeritz, T. T. Lu, J. R. Woodgett, L. B. Ivashkiv. 2006. IFN-{gamma} suppresses IL-10 production and synergizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins. Immunity 24: 563-574. [Medline]
  42. Parry, G., N. Mackman. 1997. Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-{kappa}B-mediated transcription. J. Immunol. 159: 5450-5456. [Abstract]
  43. Iwasaki, A., R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987-995. [Medline]
  44. Kaisho, T., S. Akira. 2002. Toll-like receptors as adjuvant receptors. Biochim. Biophys. Acta 1589: 1-13. [Medline]
  45. Lavelle, E. C., A. Jarnicki, E. McNeela, M. E. Armstrong, S. C. Higgins, O. Leavy, K. H. Mills. 2004. Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent. J. Leukocyte Biol. 75: 756-763. [Abstract/Free Full Text]
  46. Ryan, E. J., E. McNeela, M. Pizza, R. Rappuoli, L. O’Neill, K. H. Mills. 2000. Modulation of innate and acquired immune responses by Escherichia coli heat-labile toxin: distinct pro- and anti-inflammatory effects of the nontoxic AB complex and the enzyme activity. J. Immunol. 165: 5750-5759. [Abstract/Free Full Text]
  47. Lavelle, E. C., E. McNeela, M. E. Armstrong, O. Leavy, S. C. Higgins, K. H. Mills. 2003. Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation. J. Immunol. 171: 2384-2392. [Abstract/Free Full Text]
  48. Holmgren, J., C. Czerkinsky, K. Eriksson, A. Mharandi. 2003. Mucosal immunisation and adjuvants: a brief overview of recent advances and challenges. Vaccine 21: S89-S95. [Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
D. L. Pierce, S.-i. Nishiyama, S. Liang, M. Wang, M. Triantafilou, K. Triantafilou, F. Yoshimura, D. R. Demuth, and G. Hajishengallis
Host Adhesive Activities and Virulence of Novel Fimbrial Proteins of Porphyromonas gingivalis
Infect. Immun., August 1, 2009; 77(8): 3294 - 3301.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Liang, K. B. Hosur, S. Lu, H. F. Nawar, B. R. Weber, R. I. Tapping, T. D. Connell, and G. Hajishengallis
Mapping of a Microbial Protein Domain Involved in Binding and Activation of the TLR2/TLR1 Heterodimer
J. Immunol., March 1, 2009; 182(5): 2978 - 2985.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
C. Sundling, K. Schon, A. Morner, M. N. E. Forsell, R. T. Wyatt, R. Thorstensson, G. B. K. Hedestam, and N. Y. Lycke
CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization
J. Gen. Virol., December 1, 2008; 89(12): 2954 - 2964.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. Hajishengallis, M. Wang, S. Liang, M. Triantafilou, and K. Triantafilou
Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function
PNAS, September 9, 2008; 105(36): 13532 - 13537.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
S. Onishi, K. Honma, S. Liang, P. Stathopoulou, D. Kinane, G. Hajishengallis, and A. Sharma
Toll-Like Receptor 2-Mediated Interleukin-8 Expression in Gingival Epithelial Cells by the Tannerella forsythia Leucine-Rich Repeat Protein BspA
Infect. Immun., January 1, 2008; 76(1): 198 - 205.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liang, S.
Right arrow Articles by Hajishengallis, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liang, S.
Right arrow Articles by Hajishengallis, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS