The B Subunit of Escherichia coli Heat-Labile Enterotoxin Induces Both Caspase-Dependent and -Independent Cell Death Pathways in CD8+ T Cells

ABSTRACT The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule that acts both as an adjuvant and to stimulate immune deviation processes, resulting in the suppression of Th1-associated inflammatory responses. The ability of EtxB to alter immune reactivity is dependent on its ability to modulate immune cell function through binding to cell surface molecules, the principal receptor of which is the ubiquitous GM1-ganglioside. EtxB activates B cells and antigen-presenting cells and induces the selective apoptosis of murine CD8+ T cells. We postulated that these effects are mediated by the induction of intracellular signaling pathways following EtxB-receptor interaction. We have previously shown that CD8+ T-cell apoptosis induced by EtxB results from the activation of the transcription factor NF-κB and caspases. Here we report that while caspase activity is required for apoptosis, additional features of cell death are caspase independent. EtxB induces a rapid loss of mitochondrial membrane potential and cell viability that are unaffected by caspase inhibitors. In addition, our data suggest that these processes are independent of the activity of Bax and Bcl-2 but are mediated by nitric oxide synthase.

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The Escherichia coli heat-labile enterotoxin B subunit protects from allergic airway disease development by inducing CD4+ regulatory T cells

Mucosal Immunology ◽

10.1038/mi.2012.93 ◽

2012 ◽

Vol 6 (3) ◽

pp. 535-546 ◽

Cited By ~ 8

Author(s):

D S Donaldson ◽

M Apostolaki ◽

H K Bone ◽

C M Richards ◽

N A Williams

Keyword(s):

Escherichia Coli ◽

T Cells ◽

Regulatory T Cells ◽

Airway Disease ◽

Allergic Airway Disease ◽

Disease Development ◽

B Subunit ◽

Heat Labile ◽

Enterotoxin B

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Escherichia coli Enterotoxin B Subunit Triggers Apoptosis of CD8+ T Cells by Activating Transcription Factor c-Myc

Infection and Immunity ◽

10.1128/iai.69.8.4923-4930.2001 ◽

2001 ◽

Vol 69 (8) ◽

pp. 4923-4930 ◽

Cited By ~ 14

Author(s):

Marco Soriani ◽

Neil A. Williams ◽

Timothy R. Hirst

Keyword(s):

Escherichia Coli ◽

T Cells ◽

Transcription Factor ◽

Receptor Binding ◽

Protein Levels ◽

B Subunit ◽

Heat Labile ◽

Activating Transcription Factor ◽

Induced Apoptosis ◽

Factor C

ABSTRACT Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8+ T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8+ T cells but not of CD4+ T cells triggers the specific up-regulation of the transcription factorc-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IκBα turnover, suggesting that elevated Myc expression may be dependent on NF-κB. When CD8+ T cells were pretreated with inhibitors of IκBα turnover and NF-κB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

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Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells

Arthritis & Rheumatism ◽

10.1002/art.10328 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1671-1682 ◽

Cited By ~ 35

Author(s):

Jeffrey A. Luross ◽

Tricia Heaton ◽

Timothy R. Hirst ◽

Michael J. Day ◽

Neil A. Williams

Keyword(s):

Escherichia Coli ◽

T Cells ◽

Cd4 T Cells ◽

Autoimmune Arthritis ◽

B Subunit ◽

Heat Labile ◽

Enterotoxin B

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Mutant Escherichia coli Heat-Labile Toxin B Subunit That Separates Toxoid-Mediated Signaling and Immunomodulatory Action from Trafficking and Delivery Functions

Infection and Immunity ◽

10.1128/iai.71.3.1527-1537.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1527-1537 ◽

Cited By ~ 29

Author(s):

Sylvia A. Fraser ◽

Lolke de Haan ◽

Arron R. Hearn ◽

Heather K. Bone ◽

Robert J. Salmond ◽

...

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Nuclear Translocation ◽

Class I ◽

Receptor Interaction ◽

Cellular Interactions ◽

B Subunit ◽

Heat Labile ◽

Signaling Events ◽

B Subunits

ABSTRACT The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His→Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8+-T-cell apoptosis, (ii) activate nuclear translocation of NF-κB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits.

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Nasal Delivery of Antigen with the B Subunit of Escherichia coli Heat-Labile Enterotoxin Augments Antigen-Specific T-Cell Clonal Expansion and Differentiation

Infection and Immunity ◽

10.1128/iai.72.7.4072-4080.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 4072-4080 ◽

Cited By ~ 6

Author(s):

Maria Apostolaki ◽

Neil A. Williams

Keyword(s):

Escherichia Coli ◽

T Cells ◽

T Cell ◽

Interleukin 2 ◽

Clonal Expansion ◽

Transfer Model ◽

B Subunit ◽

Heat Labile ◽

Dividing Cells ◽

Cell Clonal Expansion

ABSTRACT Escherichia coli heat-labile enterotoxin has unique immunogenic and adjuvant properties when administered mucosally to mice. These properties have revealed the potential for its use in the development of mucosal vaccines, an area of increasing interest. However, the inherent toxicity mediated by the A subunit precludes its widespread use. This problem has led to attempts to dissociate toxicity from adjuvant function by use of the B subunit. The ability of the B subunit of E. coli heat-labile enterotoxin (EtxB) to enhance responses against antigens coadministered intranasally is demonstrated here with the use of the DO11.10 adoptive-transfer model, in which ovalbumin (OVA)-specific adoptively transferred T cells can be monitored directly by flow cytometry. Intranasal delivery of OVA with EtxB resulted in increased T-cell proliferative and systemic antibody responses against antigens. The increased Th2 cytokine production detected following in vitro restimulation of splenocyte and cervical lymph node (CLN) cells from the immunized mice correlated with increased OVA-specific immunoglobulin G1 antibody production. Flow cytometric analysis of T cells from mice early after immunization directly revealed the ability of EtxB to support antigen-specific clonal expansion and differentiation. Furthermore, while responses were first detected in the CLNs, they rapidly progressed to the spleen, where they were further sustained. Examination of CD69 expression on dividing cells supported the notion that activation induced by the presence of antigens is not sufficient to drive T-cell differentiation. Furthermore, a lack of CD25 expression on dividing cells suggested that EtxB-mediated T-cell clonal expansion may occur without a sustained requirement for interleukin 2.

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