Bacterial superantigens: provocateurs of gut dysfunction and inflammation?

Opinion

TRENDS in Immunology Vol.22 No.9 September 2001

Bacterial superantigens: provocateurs of gut dysfunction and inflammation? Derek M. McKay Inflammatory bowel diseases (IBDs) are chronic debilitating conditions, which impair the patient’s quality of life significantly. Among them, Crohn’s disease and ulcerative colitis are idiopathic disorders for which an infective etiology has long been sought. Here, we present an opinion in support of the hypothesis that bacterial superantigens can participate in the initiation, exaggeration or reactivation of enteric inflammatory disease, at least in some patients. Although the identification of a specific pathogen responsible for IBD remains a worthy pursuit, an awareness of the response to bacterial products per se will be of value in providing a comprehensive understanding of enteric pathophysiological mechanisms and their potential role in IBDs.

Bacterial superantigens (SAgs) acquired the term ‘super’ because, unlike conventional Ags, they activate ≤25% of T cells by crosslinking MHC class II molecules with a moiety on the variable portion of the β-chain of the T-cell receptor (TCR Vβ), binding outside of the Ag-specific groove of the MHC class II and TCR molecules1–3. To date, research efforts have focused largely on the immunomodulatory properties of bacterial SAgs, with the majority of reports conforming to the dogma that exposure to SAgs activates T cells, followed by anergy or depletion of the appropriate Vβ-specific T cells. At least 20 SAgs (or molecules with SAg-like activity) have been identified, and are synthesized by bacteria that occur in the gut at least sporadically (Table 1). The hypothesis presented in this article is that bacterial SAgs are potent pro-inflammatory stimuli with the potential to elicit enteropathy and cause relapses in enteric inflammatory disease. Animal studies and in vitro models

Derek M. McKay Intestinal Disease Research Programme, HSC-3N5C, 1200 Main Street West, McMaster University, Hamilton, Ontario, Canada L8N 3Z5. e-mail: mckayd@ mcmaster.ca

The idea of a bacterial etiology for inflammatory bowel diseases (IBDs) – for example, Crohn’s disease (CD) and ulcerative colitis (UC) – has always found favor among a core group of investigators and indeed, there is almost overwhelming data from animal studies and clinical observations implicating the microflora in gut inflammation4. However, despite extensive research efforts, IBDs remain idiopathic and enigmatic disorders. Is the patient with IBD responding to a http://immunology.trends.com

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nonspecific bacterial product or specific bacteria? Enterotoxins from Staphylococcus aureus [e.g. staphylococcal enterotoxin A (SEA)–SEI and toxic shock syndrome toxin (TSST)] are prototypic SAgs that cause food poisoning and toxic shock syndrome, respectively. The putative involvement of SAgs from S. aureus in enteric inflammation is not unprecedented: studies in the 1960s described a diarrheal and gut inflammatory response in animals treated with crude extracts of S. aureus or SEB (Refs 5,6). Using SEB to activate T cells, MacDonald et al. showed that the treatment of human fetal intestinal explants with SAgs resulted in hyperplasia of epithelial crypt cells and altered the ratio of crypt to surface epithelium. These events were associated with the increased production of interleukin-2 (IL-2) and interferon γ (IFN-γ)7. We have reported that SEB [or Yersinia pseudotuberculosis mitogen (YPM)] evokes a self-limiting murine enteropathy8–11. The data from these studies are summarized in Figure 1. A single lowdose treatment with SEB (i.e. 5 µg, intraperitoneal) resulted in an increased number of T cells in the lamina propria and intraepithelial compartments of the jejunum of treated mice8; thus, we speculated that exposure to SAgs could prime an individual for enhanced immune responsiveness to other Ags. Also, nitric oxide is involved in the resolution of SEB-induced abnormalities in jejunal ion transport, by the regulation of levels of IFN-γ and tumor necrosis factor α (TNF-α)11 – a finding that concurs with the studies of Florquin et al. on SAg-induced toxic shock12. Use of an in vitro coculture model comprising monolayers of the human colon-derived T84 epithelialcell line and peripheral blood mononuclear cells showed that the SEB-induced activation of immune cells resulted in increased epithelial permeability, reduced responses to pro-secretory stimuli and increased production of the T-cell and monocyte chemoattractants, regulated on activation, normal T-cell expressed and secreted (RANTES) and monocyte chemotactic protein 1 (MCP-1)13,14. These changes in epithelial function were reduced or prevented by the use of anti-IFN-γ and anti-TNF-α antibodies (Abs), or the inclusion of recombinant IL-10 or transforming growth factor β (TGF-β) in the coculture well13,15. In relation to human IBD, the data from animal and in vitro models illustrate that: (1) systemic treatment with SAgs evokes a T-cell dependent enteropathy and/or inflammation; (2) SEB-induced activation of immune cells decreases the barrier function of epithelial cells (patients with IBD have increased gut permeability, although whether this is a cause or an effect of the disease remains controversial16); and (3) the effects of SAg-mediated activation of immune cells are associated with a T helper 1 (Th1) response12,13,17, which correlates with the spectrum of cytokines observed in patients with CD (Ref. 18) and treatment with biologics (e.g. anti-TNF-αAb). Collectively, the experimental evidence shows that SAg-mediated activation of the immune system affects the form and function of the gut,

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TRENDS in Immunology Vol.22 No.9 September 2001

Table 1. Bacterial superantigensa Bacterial species

Superantigen producedb,c

Staphylococcus aureus

SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI,

Streptococcus pyrogenes

SPE-A, SPE-C, SPE-G, SPE-H, SSA, SMEZ and SMEZ-2

TSST and exfoliating toxins A and B

Yersinia pseudotuberculosis YPM YES

Clostridium perfringens

Exotoxin

Mycobacterium tuberculosis MTS aA number of viruses produce superantigens and there is fragmentary evidence of superantigens produced by protozoa53 and helminths54. bAbbreviations: MTS, Mycobacterium tuberculosis superantigen; SEA, staphylococcal enterotoxin A; SMEZ, streptococcal mitogenic exotoxin Z; SSA, streptococcal superantigen; SPE-A, streptococcal pyrogenic exotoxin A; TSST, toxic shock syndrome toxin; YES, Yersinia enterocolitica superantigen; YPM, Yersinia pseudotuberculosis mitogen. cSee Refs 1,2.

and supports the hypothesis that these bacterial products can initiate or exacerbate a secretory or inflammatory enteropathy. The question remains whether exposure to SAgs has any significance for human gut dysfunction or disease? Human studies

The evidence that SAgs are etiological agents in inflammation is strongest for psoriasis, for which an (a)

(b)

(c) Jejunum Colon

Baseline Isc ∆Isc to ETS ∆Isc to CCH

NT

∆Isc to FSK ∆Isc to PGE2

NT

–

Crypt depth

Administration of SEB or YPM (i.p.)

Minor

No

Yes

NT

NT

Number of T cells and IELs Activation of LPLs Serum level of IL-2, IFN-γ and TNF-α Neutrophil infiltrates

No

No NT

Gut MHC II expression Gut iNOS induction

120

Yes

NT

4h 48 h

80

* * *

40

*

* *

0 0

–

Villus height

Tissue damage

∆Isc to CCH (µA cm–2)

Yersinia enterocolitica

association with HLA-DR has been noted19,20, and multi-system vasculitis (Kawasaki disease), for which a Yersinia SAg has been implicated19. Data have suggested a role for SAgs in arthritis, insulindependent diabetes and Celiac disease also (by molecular mimicry in the case of Celiac disease)21–23. Indirect evidence for the involvement of SAgs in IBD comes from the analysis of blood- and gut-derived lymphocytes that have skewed expression of TCR Vβ in cohorts of patients with CD (Refs 24,25). Certainly, SAgs are able to activate gut-derived immune cells26. However, the clonal accumulation of T cells in the lesions of patients with CD has also been seen in experiments where the investigators rejected the involvement of SAgs (Ref. 27). Skewed expression of TCR Vβ is used as evidence of exposure to SAgs (Refs 20–25), but is this always the case? Mycoplasma arthritidis mitogen (MAM) was found to bind to the complementarity determining region 3 (CDR3) of the TCR, leading the authors to speculate that some SAgs might utilize a restricted TCR repertoire and therefore, not leave the tell-tale ‘Vβ footprint’ typical of staphylococcal SAgs (Ref. 28). If other nonstaphylococcal, nonstreptococcal SAgs act similarly to MAM then the intriguing, but perplexing, scenario that the analysis of TCR Vβ will be insufficient to

1

5

50

100

SEB (i.p. µg mouse–1)

(d) Serum IFN-γ (ng ml–1)

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*

10 8

*

6 4 2 0

ND BALB/c BALB/c SCID SCID rec.SCID + SEB + SEB + SEB TRENDS in Immunology

Fig. 1. (a) Systemic treatment of Balb/c male mice with intraperitoneal (i.p.) injection of Staphylococcus aureus enterotoxin B (SEB; 5–100 µg) or Yersinia pseudotuberculosis mitogen (YPM; 20 µg) leads to (b) a selflimiting enteropathy characterized by altered jejunal and colonic form and/or function. (c) The SEB dose-dependent diminution of the Isc response to CCH (n = 4) of jejunal tissue mounted in Ussing chambers four hours (4 h) and 48 h after i.p. injection of the superantigen (SAg). The SAg-induced enteropathy was not observed in mice lacking thymusdependent T cells [i.e. severe combined immunodeficient (SCID) mice] but could be evoked in SCID mice reconstituted with CD4+ T cells. (d) The increased serum level of interferon γ (IFN-γ) observed 4 h after treatment of Balb/c mice with SEB (5 µg, i.p.) did not occur in SCID mice, but was

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recapitulated in the SCID mice reconstituted with CD4+ T cells (rec.SCID; n = 4). Abbreviations: CCH, carbachol at 10−4 M (evokes Ca2+-mediated secretion of Cl−); ETS, electrical transmural nerve stimulation; FSK, forskolin at 10−5 M [evokes cyclic adenosine monophosphate (cAMP)-mediated secretion of Cl−]; IELs, intraepithelial lymphocytes; IL-2, interleukin-2; iNOS, inducible nitric oxide synthase; Isc, short-circuit current (indicates the net active electrolyte flux that creates the driving force for directed water movement); LPLs, lamina propria lymphocytes; ND, not detected; NT, not tested; PGE2, prostaglandin E2 at 10−5 M (evokes cAMP-mediated secretion of Cl−); TNF-α, tumor necrosis factor α. Symbols: *, significantly different from control data (P <0.05)8–11; ∆, change in.

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implicate or refute the involvement of SAgs in disease must be considered. Thus, in light of the current clinical data, it is naive to suggest that SAgs are the universal cause of CD. In presenting the postulate that these T-cell stimuli are relevant to the pathogenesis of a cohort(s) of patients with IBD, a number of issues arise. Can SAgs be identified in IBD?

The short answer is that a SAg responsible for IBD has not been identified. However, the histopathological similarity of Jonge’s disease in cattle (caused by mycobacterial infection) and human ileal yersiniosis with CD has not gone unnoticed, and species from both genera can produce SAgs (Table 1). The notion that Mycobacterium paratuberculosis is the cause of CD is controversial, with the balance of data refuting rather than supporting this hypothesis. However, the existence of SAgs derived from M. tuberculosis29 allows for the possibility that a SAg might be relevant to the disease process in patients with CD and confirmed mycobacterial infection30. A bacterial DNA sequence, designated I2, has been identified in 43% of the colonic lesions from a sample of patients with CD (Ref. 31). Recently, the same investigators presented preliminary data indicating that the I2 protein (from Pseudomonas fluorescens) is a novel SAg, activating naive and memory CD4+ Vβ5.1+ and Vβ5.2+ T cells32. These tantalizing observations are the first demonstration of a SAg involved in IBD, but require confirmation from other patient samples and by other investigators. Do SAgs synergize with other bacterial products to cause IBD?

If SAgs elicit the activation of T cells followed by their depletion, why are increased numbers of specific TCR Vβ+ T cells observed in some patients with CD? Either these patients have been exposed to SAgs recently or, as Marrack et al. proposed, the depletion of T cells can be negated by exposure to other stimuli, such as lipopolysaccharide (LPS)-induced TNF-α (Ref. 33) [levels of TNF-α are increased in CD (Ref. 18)]. Indeed, these researchers reported increased numbers of murine CD4+ Vβ3+ T cells 95 days after the coadministration of SEA and LPS (Ref. 34). Also, the induction of expression of OX40 on murine CD4+ T cells after cotreatment with SEA and LPS has been postulated to be a cognate signal that overcomes the depletion of T cells characteristic of exposure to SAg only, which enhances the survival of memory T cells35. Often, the expression of OX40 and OX40 ligand is increased in the lamina propria of patients with IBD (Ref. 36). Furthermore, mice treated with SEB and LPS have increased numbers of apoptotic enteric epithelial cells37, and a role for apoptosis in UC has been suggested38. Given the enormous load of Gram-negative enteric bacteria, one would accept intuitively that SAgs and LPS could gain access to the lamina propria simultaneously, where they would act in concert to evoke altered gut function, an acute http://immunology.trends.com

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inflammatory response and potentially, chronic inflammatory disease. The combinatorial effect of enterotoxins from S. aureus and other bacterial products could be relevant to the pathophysiology of IBD also. For example, Pseudomonas aeruginosa exotoxin A and SEB increase and prolong elevated murine serum levels of TNF-α synergistically39. Also, bacterial cell wall peptidoglycan–polysaccharides (PG–PSs) have been implicated in IBD (Ref. 40) but a cooperative effect of PG-PSs and SAgs in eliciting gut dysfunction and/or inflammation has not been tested as yet. Is there a role for SAgs in IBD through an association with HLA?

A genetic predisposition to IBD is being defined. Studies assessing various ethnic populations of patients with CD and UC have provided evidence correlating the expression of HLA (particularly HLA-DR subtypes) with IBD (Refs 41,42). Generally, the activity of SAgs is MHC-class-II-dependent and interestingly, for those SAgs for which binding to MHC class II has been examined (e.g. S. aureus enterotoxins), the interaction is with HLA-DR1 (Ref. 3). This presents the possibility, although untested, that the triggering of IBD by SAgs could be mediated by a genetic predisposition to express certain subtypes of HLA, as has been postulated for psoriasis20. A more speculative supposition, but which converges on a genetic predisposition hypothesis also, is that individuals with increased numbers of certain TCR Vβ expression profiles might be at greater risk of SAg-induced enteropathy. Figure 2 illustrates the means by which exposure to SAgs could elicit or modulate IBD. Although an association between HLA and UC might exist, there is less evidence in support of a microbial etiology for UC compared with CD. [However, it is not unusual for patients to present with UC after an infectious episode (S.M. Collins, pers. commun.).] In UC, the presence of autoAbs and a Th2-skewed cytokine profile suggests that this might be an autoimmune condition. Here again, SAgs can act as initiating or predisposing factors. The SAg-induced polyclonal proliferation of T cells would increase the number of autoreactive effector T cells, which during traffic through the circulation and tissues, encounter the appropriate TCR-specific Ag and are activated43,44. Can SAgs reactivate colitis?

The insidious relapsing nature of IBD is of considerable concern to both the patient and medical practitioner. A number of mechanisms have been proposed to elicit relapse in IBD, including infection. Animal models of arthritis, allergic encephalitis and nephritis have all shown that these disorders can be reactivated or exaggerated by treatment with SAgs (Refs 45–47). Similar data are not available for gut inflammation. Recently, we generated preliminary data showing that the direct introduction of SEB into the colonic lumen of mice recovering from colitis

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Fig. 2. Hypothetical flowdiagram models illustrating the potential role(s) for bacterial superantigens (SAgs) in (a) the initiation of or (b) the contribution to enteric inflammation. Abbreviations: CD, Crohn’s disease; NSAI, nonsteroidal antiinflammatory; TCR Vβ, T-cell receptor variable region β; UC, ulcerative colitis.

Opinion

(a)

TRENDS in Immunology Vol.22 No.9 September 2001

(b)

SAg causes colitis SAg exposure: • systemic • intraluminal

Genetic predisposition e.g. HLA association (TCR Vβ expression?)

Self-limiting enteropathy or inflammation

Bacterial products, pro-inflammatory cytokines and T-cell survival stimuli

CD

Priming

Reactivation

SAg exposure

History of colitis

Individual primed for disease

Expansion of autoreactive T cells

T cells find autoantigen

CD

Contributory effect of SAg

Sub-threshold pro-inflammatory stimulus

Enteric inflammatory disease

SAg exposure

Other factors: • stress • use of NSAI drugs

Induction of relapse of colitis

UC TRENDS in Immunology

induced by dextran sulfate sodium (DSS) reactivated the disease or worsened the inflammation48. Moreover, and in line with anecdotal clinical observations, mice that lost >10% of their body weight in response to DSS had a greater propensity for the reactivation of disease by SAgs. An intriguing scenario can be envisaged, in which the disease can be reactivated by a mechanism – in this instance, SAg-driven activation of the immune system – that has little or nothing to do with the etiology of disease. Further characterization of this murine system might provide a model in which initial disease severity can be used as a predictor of susceptibility to relapse or reactivation of disease elicited by infection or bacterial products. Non-T-cell effects of SAgs

Acknowledgments Citations from the author's laboratory were funded by the Crohn's and Colitis Foundation of Canada and the Canadian Institutes for Health Research (formerly Medical Research Council of Canada).

Macrophages have been implicated in IBD, and fibroblasts are able to modulate the enteric inflammatory response also49; both cell types can act as Ag-presenting cells (APCs) for SAgs. Furthermore, in the absence of T cells, SAgs with two MHC class IIbinding domains (i.e. SEA and SED) have been found to activate MHC class II+ fibroblasts and macrophagecell lines directly50,51. Also, epithelial-cell lines treated with IFN-γ (which induces the expression of MHC class II) or freshly isolated enterocytes can act as APCs for SAgs and drive the proliferation of T cells52, but a direct effect of SAg on epithelial function has not been determined. Thus, under physiological conditions it seems improbable that SAgs would have appreciable effects on stromal cells that have little constitutive expression of MHC class II. However, direct effects on tissue-resident dendritic cells are a distinct, but http://immunology.trends.com

untested possibility. By contrast, under inflammatory conditions, when the expression of MHC class II is upregulated significantly on many cell types, the environment would be rich in receptors for SAgs, which would allow for the direct activation of stromal cells and APCs that could affect the progression and resolution of enteric inflammation. Therefore, in an extension of the canonical view of SAgs as stimuli for T cells, it seems reasonable to suggest that SAg–APC interactions could contribute to IBD. Conclusions

Undoubtedly, the gut microflora are involved in enteric inflammatory disease4. However, the identity of a pathogen responsible for IBD has not been forthcoming. Although it would be folly to abandon the search for a specific pathogen, particularly in view of the role of Helicobacter pylori in gastric ulceration, we contend that, given the right circumstances, bacterial products from any one of a multitude of organisms are sufficient to cause inflammatory disease. By virtue of their ability to activate large numbers of T cells and perhaps also APCs (including MHC class II+ stromal cells), SAgs are a major proinflammatory stimulus, which, either alone or in combination with other bacterial products (e.g. LPS), have the capacity to prime an individual for the development of colitis, initiate inflammatory disease or evoke relapses in IBD. It is hoped that the data and issues presented here will provoke debate and research to test the suppositions that have been raised. If one accepts that IBDs are in fact heterologous diseases, then a broadening of research

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initiatives to consider the pathogenicity of bacterial products per se (individually and in concert) will contribute to the definition of enteric References 1 Fraser, J. et al. (2000) Superantigens – powerful modifiers of the immune system. Mol. Med. Today 6, 125–132 2 Lavoie, P.M. et al. (1999) Understanding the mechanism of action of bacterial superantigens from a decade of research. Immunol. Rev. 168, 257–269 3 Li, H. et al. (1999) The structural basis of T-cell activation by superantigens. Annu. Rev. Immunol. 17, 435–466 4 McKay, D.M. (1999) Intestinal inflammation and the gut microflora. Can. J. Gastroenterol. 13, 509–516 5 Kent, T.H. (1965) Staphylococcal enterotoxin gastroenteritis in rhesus monkeys. Am. J. Pathol. 48, 387–407 6 van Prohaska, J. (1963) Role of staphylococcal enterotoxin in the induction of experimental ileitis. Ann. Surg. 158, 492–497 7 Lionetti, P. et al. (1993) Activation of mucosal Vβ3+ T cells and tissue damage in human small intestine by the bacterial superantigen, Staphylococcus aureus enterotoxin B. Eur. J. Immunol. 23, 664–668 8 Donnelly, G.A.E. et al. (1999) Colonic epithelial physiology is altered in response to the bacterial superantigen Yersinia pseudotuberculosis mitogen (YPM). J. Infect. Dis. 180, 1590–1596 9 Benjamin, M.A. et al. (1998) Changes in murine jejunal morphology evoked by the bacterial superantigen Staphylococcus aureus enterotoxin B are mediated by CD4+ T cells. Infect. Immun. 66, 2193–2199 10 McKay, D.M. et al. (1998) CD4+ T cells mediate superantigen-induced abnormalities in murine jejunal ion transport. Am. J. Physiol. 275, G29–G38 11 McKay, D.M. et al. (1999) Nitric oxide participates in the recovery of normal jejunal epithelial ion transport following exposure to the superantigen Staphylococcus aureus enterotoxin B. J. Immunol. 163, 4519–4526 12 Florquin, S. et al. (1994) The protective role of endogenously synthesized nitric oxide in staphylococcal enterotoxin-B-induced shock in mice. J. Exp. Med. 180, 1153–1158 13 McKay, D.M. and Singh, P.K. (1997) Superantigen-activation of immune cells evokes epithelial (T84) transport and barrier abnormalities via interferon γ and tumour necrosis factor α. Inhibition of increased permeability, but not diminished secretory responses by transforming growth factor β2. J. Immunol. 159, 2382–2390 14 Jedrzkiewicz, S. et al. (1999) Superantigen immune stimulation evokes epithelial monocyte chemoattractant protein 1 and RANTES production. Infect. Immun. 67, 6198–6202 15 Lu, J. et al. (1998) Epithelial transport and barrier abnormalities evoked by superantigenactivated immune cells are inhibited by IL-10, but not IL-4. J. Pharmacol. Exp. Ther. 287, 128–136 16 Hollander, D. (1992) The intestinal barrier: a hypothesis as to its regulation and involvement in Crohn’s disease. Scand. J. Gastroenterol. 27, 721–726 17 Bette, M. et al. (1993) Distribution and kinetics of superantigen-induced cytokine expression in mouse spleen. J. Exp. Med. 178, 1531–1540

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