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pathogen interactions at mucosal surfaces: Immune consequences
Research in Microbiology 153 (2002) 455–459 www.elsevier.com/locate/resmic
Host/pathogen interactions at mucosal surfaces: Immune consequences Simon Clare, Alan Huett, Gordon Dougan ∗ Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, UK Received 5 June 2002; accepted 11 June 2002
Abstract The mucosal immune system has evolved to protect the host against the establishment of infections at or through the mucosal surfaces of the body. Protective immunity must be activated to specific pathogenic agents or their products but inappropriate immune responses to food/environmental antigens must be avoided. Thus, the mucosal immune system is under tight regulation. Pathogenic bacteria and their products can be exploited as specific probes of mucosal immune responses. Bacterial enterotoxins such as cholera toxin are potent mucosal immunogens and adjuvants that activate both mucosal and systemic immune responses. Infection models involving microorganisms such as Citrobacter rodentium can also be used to investigate the consequences of mucosal colonisation that lead to immune disfunction. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Mucosal adjuvant; Enterotoxin; Infection; Citrobacter rodentium
1. Introduction The mucosal surfaces of the body are the sites through which mammals exchange the nutrients and gases essential for survival. As a consequence of these physiological demands and a requirement for exposure to the environment, eucaryotic cells associated with these surfaces are vulnerable to attack by potential microbial pathogens. Microbes have evolved mechanisms for attaching to and invading mucosal surfaces, whereas the host immune system has evolved to counter these threats. This constant battle between the host and the pathogen has fine tuned the immune system and tight regulatory mechanisms are in place to prevent inappropriate responses that could lead to immune pathology [8]. This is particularly important as the intestine of mammals is heavily colonised by commensal bacteria that continuously release antigens, which may subsequently interact with gut-associated immune cells. The mammalian immune system shows partial compartmentalisation into the systemic and mucosal systems [23]. The mucosal system is associated with local immune responses and has associated with it, specialised lymphoid tissues known * Correspondence and reprints.
E-mail address: [email protected] (G. Dougan).
as gut-associated lymphoid tissue (GALT) in the gut and the equivalent bronchial-associated lymphoid tissues (BALT) in the respiratory tract [18]. Pathogen-associated immune responses in the GALT and BALT drive potentially protective IgA production and local cellular responses. Immune responses at these sites have to have some capacity to discriminate between food/environmental antigens, which are non-threatening, and antigens from pathogens with the potential to cause damage and disease. The default mode at mature mucosal surfaces is to be non-responsive (in terms of the production of IgA and activation of effector mechanisms) and this factor limits immune responses to most antigens encountered at mucosal surfaces. However, it is essential that appropriate protective immunity, both innate and acquired, is rapidly stimulated against the pathogenic threat. At the moment, little is known about how the mucosal immune system regulates this balance. We do know that infection with many pathogens stimulates rapid mucosal and sometimes systemic responses that can lead to protection. We also know that certain products from microorganisms can activate immunity through interaction with specific receptors such as members of the Toll receptor family [22]. Toll receptors recognise generic molecules specific for microbes such as unmodified DNA, lipopolysaccharide and flagella. We also know that some microbial molecules that actively bind to
0923-2508/02/$ – see front matter 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 1 3 4 5 - 1
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surface molecules on eucaryotic cells, such as toxins and fimbriae, are capable of acting as immunogens at mucosal surfaces [3]. With some antigens such as pertussis toxin or filamentous haemagglutinin from Bordetella pertussis, inactivation of cell binding activity has been shown to dramatically reduce mucosal but not systemic immunogenicity [6]. Thus, the mucosal immune system may be activated via targeted interactions involving pathogen products and immune cells.
2. Enterotoxins as mucosal immunogens and adjuvants Perhaps the best known examples of pathogen-derived mucosal immunogens are members of the cholera enterotoxin group typified by cholera toxin (CT) and the heat-labile enterotoxins (LT) of Escherichia coli. Microgram quantities of these toxins can stimulate potent mucosal and systemic antitoxin immune responses in a number of animals. Indeed, in some species these are so powerful that immune responses can also be stimulated to particular co-administered antigens that are not normally mucosal immunogens [28]. In other words CT and LT have potent mucosal adjuvant properties. Many studies on LT and CT have progressed in murine systems as these animals, unlike humans, have good tolerance to the toxicity of these molecules and immunisations can be conducted in the absence of significant side effects. Many murine studies have investigated the mucosal adjuvant activities of CT and LT using different bystander antigens and similar investigations are now being conducted in other species [35]. CT and LT are typical AB type toxins and share significant homology in terms of their primary and secondary structures. The A subunit of both toxins possesses ADPribosyltransferase activity that is responsible for the toxicity of the molecule. The A subunit is usually found tightly associated with the B subunit which is a pentemeric protein composed of five small identical polypeptides. The structure of both toxins have been solved using X-ray crystallography and they confirm that the toxins are very highly related [31]. The B subunit is the eucaryotic cell targeting domain and binds to non-protein receptors. CT has a high affinity for GM1 gangliosides, which are found in abundance on many eucaryotic cell types [16], whereas LTs are apparently more promiscuous in that in addition to GM1ganglioside they also bind to the glyco component of some glycoproteins [33]. After interaction of the B subunit with target cells the holotoxin enters endosomes and is transferred to the Golgi where the two subunits separate. The A subunit is directed in a retrograde manner through the Golgi where it eventually ends up in the cytosol. At this point the enzymatic activity of the toxin is expressed and this leads to the modification of host G proteins leading to the accumulation of cAMP in target cells. CT and LT can stimulate the production of secretory IgA, serum antibodies and potent cellular responses to co-
administered protein and in many infection models, protection, as has been demonstrated following CT or LT-based mucosal (oral or nasal) immunisation [17]. Significantly LT and CT can stimulate both CD4 and CD8-associated cellular responses [11,25]. The CD8 responses have been shown to be associated with cytotoxic activity mediating protection against viral infection [30]. Strenuous efforts have been made to decipher the mechanisms involved in the mucosal adjuvant activity of these proteins. The type of immune response associated with these toxins as adjuvants has been thoroughly described although the molecular basis of the activity remains elusive. Indeed, the toxins are likely to have pleiotrophic effects on both immune and non-immune eucaryotic cells complicating the interpretation of investigations [12]. A key question about the practical use of these toxins has been whether toxin activity can be separated from the adjuvant activities of the enterotoxins. Work with site-directed mutants of the toxin that inactivate ADP-ribosyltransferase activity but that do not significantly affect the stablility or assembly of the holotoxin has clearly shown that toxicity and adjuvant activity can be at least partially separated [10,11, 25]. LT mutants such as LTK63 (with an inactivating lysine amino acid substitution at position 63 of the A subunit) have now been well characterised in many different laboratories and they clearly retain adjuvant activity. It is also clear that the enzymatically active A domain also contributes, possibly in several different ways, to adjuvant activity [2,12].
3. Murine Citrobacter rodentium infection associated with mucosal immune disregulation Certain bacterial infection models also offer an opportunity to investigate the immune regulatory mechanisms operating during a live infection as well as the impact of immune disregulation on gut physiology. The murine pathogen C. rodentium possesses several features, which make it ideally suited to model both pathogenic E. coli infections and the immune mechanisms underpinning inflammatory bowel disease. Infection with C. rodentium is characterised by colonic hyperplasia primarily at the distal end of the colon and spreading up towards the caecum with increasing severity of infection [4]. The colon becomes swollen and more rigid as hyperplasia reaches a peak around day 14 postinfection. Wild-type animals resolve infection by day 28 and are resistant to rechallenge. The increase in colon size and weight can be attributed to cellular infiltration into the crypts and lamina propria as well as enterocyte proliferation. In severe infection the faeces become soft and in rare cases further complications, such as rectal prolapse, may ensue [19]. Immunostaining to localise bacteria reveals that in most wildtype infections C. rodentium is confined to the apical surfaces of the enterocytes. In immunocompromised mice and severe infections the pathogen appears to colonise deeper into the crypts as well as more extensively over the epithe-
S. Clare et al. / Research in Microbiology 153 (2002) 455–459
lial surface. This suggests host immune mechanisms play an important role in limiting infection to the mucosal epithelial surface and protecting deeper tissues. The mechanisms responsible for control are unknown and may depend upon a number of innate immune responses as well as physical factors such as mucus secretion and epithelial shedding. An important virulence-associated determinant of C. rodentium is the locus of enterocyte effacement (LEE), a 35-kb pathogenicity island with high homology to similar determinants encoded by both enterohaemorragic and enteropathogenic E. coli (EHEC and EPEC, respectively) [9, 21]. LEE-positive C. rodentium form characteristic pedestallike structures on the murine enterocyte surface known as attaching/effacing (A/E) lesions. The A/E lesions formed in mice are indistinguishable in morphology from those of EPEC on human enterocytes. A functional LEE is necessary and sufficient for bacteria to produce A/E lesions on epithelial cells [21]. Significantly, C. rodentium that lack LEE do not colonise the murine intestine to a significant level and do not induce colonic hyperplasia [27]. Like several other pathogenicity islands from intestinal pathogens, the LEE encodes a type three secretion system and associated effector proteins. The LEE-encoded apparatus allows secretion of bacterial effectors directly into host enterocytes to subvert normal epithelial (and possibly immune) function and allow intimate attachment of the bacterium to the host mucosa. Initial epithelial contact of C. rodentium with enterocytes leads to destruction (effacement) of the brush border microvilli. This is followed by translocation of the intimin receptor (Tir) and strong attachment of the bacterium via the intimin/Tir interaction. During this process the host cytoskeleton undergoes dramatic rearrangement leading to the formation of the characteristic pedestal of the A/E lesion. Experimental investigations of these critical pathogen/host mucosal interactions in vitro have been limited by the inability of simple cell culture to mimic the complex morphology and host reactions involved in the gut. The use of ex vivo explants from human biopsy samples and a murine model using C. rodentium have allowed substantial strides to be made both in molecular characterisation of the host-pathogen interaction and the immune mechanisms involved [29]. Attempts are now being made to understand the immune responses stimulated by C. rodentium infection and the mechanisms involved in protection and hyperplasia. Innate immune responses, inducing induction of antimicrobial peptides (defensins) can be detected during C. rodentium infection [29]. The localised production of nitric oxide by inducible nitric oxide synthase (INOS) can be clearly observed by immunohistochemistry, although it appears to play a minor role in immunity, since INOS-/-mice are no more susceptible to C. rodentium than wild-type [29]. This is distinct from a systemic infection such as Salmonella typhimurium where lack of INOS is almost always fatal [20]. Other innate immune mechanisms are less well studied in this model. For example the Toll-like receptors (TLRs) are likely to play
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an important role in macrophage, neutrophil and epithelial activation. The downregulation of TLR4 on epithelial cells, gut macrophages and T cells serves to limit the inappropriate response to commensal lipopolysaccharide [5,24] and this may be true of other molecules responding to bacterial components [32]. Whether the recruitment of immune cells to the lamina propria from systemic circulation leads to a breakdown in this tolerance and TLR activation is not known, but may be important for the strong Th1 response observed following C. rodentium infection [13–15,34]. Whilst TLR4 is unlikely to be the entire story mice lacking functional signalling from this molecule, e.g. C3HEJ, show increased susceptibility, higher colonic pathogen burden and greater hyperplasia when challenged with C. rodentium [4]. There has been much progress in recent years identifying the host immune mechanisms required for control. Many of the infiltrating cells are CD4+ and the cytokine milieu suggests a strongly polarised Th1 response [14,15]. Both interferon γ(IFN-γ) and interleukin 12 (IL-12) have been implicated in host defence against C. rodentium. IL-12−/− mice exhibit higher colonic and systemic bacterial counts and some succumb to systemic infections. Surviving animals take two weeks longer to clear infection than wild types. IFN-γ−/− animals also support higher pathogen loads and take approximately seven days longer to clear infection than wild-type [29]. The role of TNF-α in Crohn’s disease and other forms of inflammatory bowel disease is well documented and antiTNF-α therapy is often effective in ameliorating symptoms [1,7,26]. Whilst in C. rodentium infections TNF-α is not required for clearance, mice lacking TNF-α receptor show higher bacterial burdens, increased IL-12 production and greater hyperplasia, but resolve infection as rapidly as wild types [13]. The fact that lack of the TNF-α receptor induces greater hyperplasia suggests that TNF-α may modulate the Th1 mucosal response and serve to limit pathology in wild-type animals. Studies in RAG1 knockout mice (lacking a T- and B-cell repertoire) have revealed that these cells are required for elimination of the infection. These mice fail to clear the bacteria and often succumb to infection [34]. CD4+ -cell-depleted mice fail to clear infection and antiC. rodentium serum antibody titres are significantly reduced compared to nondepleted animals (Simmons et al., in press). Depletion of CD8+ cells has no significant effect upon clearance or pathology. The role of B cells has been further clarified by the use of Igh6−/− mice (B-cell-deficient) (Simmons et al., in press). These animals remain hyperplastic and fail to resolve infection until at least day 56 postinfection, although they do not succumb to systemic disease. Thus it would appear that antibody production is required for clearance of C. rodentium and CD4+ T-cell help is needed to produce a resolving level of antibody.
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4. Conclusions As we learn more about the molecular mechanisms underpinning the pathogenicity and immunology of mucosal bacterial infections new opportunities are emerging to generate novel therapeutic approaches. Bacteria activate the mucosal and systemic immune systems through specific cues that allow immune cells to identify their presence. The location of bacterial antigens within the context of the mucosal surfaces may be critical and the ability of products to interact actively with immune cells may stimulate active immune responses. Bacterial products such as enterotoxins will continue to be used as useful probes of the mucosal immune system and further studies on pathogens such as C. rodentium, that induce immune pathology at mucosal surfaces, will be used to model conditions such as inflammatory bowel disease and provide routes towards therapies.
Acknowledgements This work is supported by The Wellcome Trust.
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[12] M.M. Giuliani, G. Del Giudice, V. Giannelli, G. Dougan, G. Douce, R. Rappuoli, M. Pizza, Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP- ribosyltransferase activity, J. Exp. Med. 187 (1998) 1123–1132. [13] N.S. Goncalves, M. Ghaem-Maghami, G. Monteleone, G. Frankel, G. Dougan, D.J.M. Lewis, C.P. Simmons, T.T. MacDonald, Critical role for tumor necrosis factor alpha in controlling the number of lumenal pathogenic bacteria and immunopathology of infectious colitis, Infect. Immun. 69 (2001) 6651–6659. [14] L.M. Higgins, G. Frankel, I. Connerton, N.S. Goncalves, G. Dougan, T.T. McDonald, Role of bacterial intimin in colonic hyperplasia and inflammation, Science 285 (1999) 588–591. [15] L.M. Higgins, G. Frankel, G. Douce, G. Dougan, T.T. MacDonald, Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to murine inflammatory bowel disease, Infect. Immun. 67 (1999) 3031–3039. [16] J. Holmgren, I. Lonnroth, A.L. Svennerholm, Fixation and inactivation of cholera toxin by GM1 ganglioside, Scand. J. Infect. Dis. 5 (1973) 77–78. [17] H. Jakobsen, D. Schulz, M. Pizza, R. Rappuoli, I. Jonsdottir, Intranasal immunisation with pneumococcal polysaccharide conjugate vaccines with nontoxic mutants of Escherichia coli heat-labile enterotoxins as adjuvants protects mice against invasive pneumococcal infections, Infect. Immun. 67 (1999) 5892–5897. [18] M.M. Levine, G. Dougan, Optimism over vaccines administered through mucosal surfaces, Lancet 351 (1998) 1375–1376. [19] S.A. Luperchio, D.B. Schauer, Molecular pathogenesis of Citrobacter rodentium transmissible murine colonic hyperplasia, Microbes Infect. 3 (2001) 333–340. [20] P. Mastroeni, A. Vazquez-Torres, F. Fang, Y. Xu, S. Khan, C.E. Hormaeche, G. Dougan, Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II Effects on microbial proliferation and host survival in vivo, J. Exp. Med. 192 (2000) 237–247. [21] T.K. McDaniel, J.B. Kaper, A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12, Mol. Microbiol. 23 (1997) 399–407. [22] R. Medzhitov, Toll-like receptors and innate immunity, Nature Rev. Immunol. 1 (2001) 135–145. [23] C. Nagler-Anderson, Man the barrier! Strategic defences in the intestinal mucosa, Nature Rev. Immunol. 1 (2001) 59–67. [24] S. Naik, E.J. Kelly, L. Meijer, S. Petterson, I.R. Sanderson, Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium, J. Pediatr. Gastroenterol. Nutr. 32 (2001) 449– 453. [25] R. Rappuoli, M. Pizza, G. Douce, G. Dougan, Structure and mucosal adjuvanticity of cholera and Escherichia coli heat-labile enterotoxins, Immunol. Today 20 (1999) 493–500. [26] W.J. Sandborn, B.G. Feagan, S.B. Hanauer, D.H. Present, L.R. Sutherland, M.A. Kamm, D.C. Wolf, J.P. Baker, C. Hawkey, A. Archambault, C.N. Bernstein, C. Novak, P.K. Heath, S.R. Targan, An engineered human antibody to TNF (CDP571) for active Crohn’s disease: A randomized double-blind placebo-controlled trial, Gastroenterology 120 (2001) 1330–1338. [27] D.B. Schauer, S. Falkow, The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia, Infect. Immun. 61 (1993) 4654–4661. [28] C.P. Simmons, M. Ghaem-Magami, L. Petrovska, L. Lopes, B.M. Chain, N.A. Williams, G. Dougan, Immunomodulation using bacterial enterotoxins, Scand. J. Immunol. 53 (2001) 218–226. [29] C.P. Simmons, N.S. Goncalves, M. Ghaem-Maghami, M. BajajElliott, S. Clare, B. Neves, G. Frankel, G. Dougan, T.T. MacDonald, Impaired resistance and enhanced pathology during infection with a non-invasive, attaching-effacing enteric bacterial pathogen Citrobacter rodentium, in mice lacking IL-12 or INFγ, J. Immunol. 168 (2002) 1804–1812.
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[30] C.P. Simmons, T. Hussell, T. Sparer, G. Walzl, P. Openshaw, G. Dougan, Mucosally elicited CD8 T cell responses mediate resistance to Respiritory Syncytial Virus infection independently of IFNγ but contribute to immune-associated weight loss, J. Immunol. 166 (2001) 1106–1113. [31] T.K. Sixma, K.H. Kalk, B.A. Van Zanten, Z. Dauter, J. Kingma, B. Witholt, W.G.J. Hol, Refined structure of Escherichia coli heatlabile enterotoxin, a close relative of cholera toxin, J. Mol. Biol. 230 (1993) 890–918. [32] P.D. Smith, L.E. Smythies, M. Mosteller-Barnum, D.A. Sibley, M.W. Russell, M. Merger, M.T. Sellers, J.M. Orenstein, T. Shimada, M.F. Graham, H. Kubagawa, Intestinal macrophages lack CD14 and CD89 and consequently are down-regulated for LPS- and IgA-mediated activities, J. Immunol. 167 (2001) 2651–2656.
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[33] S. Teneberg, T.R. Hirst, J. Angstrom, K.A. Karlsson, Comparison of the glycolipid-binding specificities of cholera toxin and porcine Escherichia coli heat-labile enterotoxin: Identification of a receptoractive non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine, Glycoconj. J. 11 (1994) 533–540. [34] B.A. Vallance, W. Deng, L.A. Knodler, B.B. Finlay, Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection, Infect. Immun. 70 (2002) 2070–2081. [35] W. Van den Broeck, E. Cox, B.M. Goddeeris, Induction of immune responses in pigs following oral administration of purified F4 fimbriae, Vaccine 10 (1999) 2020–2029.
Host/pathogen interactions at mucosal surfaces: Immune consequences Simon Clare, Alan Huett, Gordon Dougan ∗ Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, UK Received 5 June 2002; accepted 11 June 2002
Abstract The mucosal immune system has evolved to protect the host against the establishment of infections at or through the mucosal surfaces of the body. Protective immunity must be activated to specific pathogenic agents or their products but inappropriate immune responses to food/environmental antigens must be avoided. Thus, the mucosal immune system is under tight regulation. Pathogenic bacteria and their products can be exploited as specific probes of mucosal immune responses. Bacterial enterotoxins such as cholera toxin are potent mucosal immunogens and adjuvants that activate both mucosal and systemic immune responses. Infection models involving microorganisms such as Citrobacter rodentium can also be used to investigate the consequences of mucosal colonisation that lead to immune disfunction. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Mucosal adjuvant; Enterotoxin; Infection; Citrobacter rodentium
1. Introduction The mucosal surfaces of the body are the sites through which mammals exchange the nutrients and gases essential for survival. As a consequence of these physiological demands and a requirement for exposure to the environment, eucaryotic cells associated with these surfaces are vulnerable to attack by potential microbial pathogens. Microbes have evolved mechanisms for attaching to and invading mucosal surfaces, whereas the host immune system has evolved to counter these threats. This constant battle between the host and the pathogen has fine tuned the immune system and tight regulatory mechanisms are in place to prevent inappropriate responses that could lead to immune pathology [8]. This is particularly important as the intestine of mammals is heavily colonised by commensal bacteria that continuously release antigens, which may subsequently interact with gut-associated immune cells. The mammalian immune system shows partial compartmentalisation into the systemic and mucosal systems [23]. The mucosal system is associated with local immune responses and has associated with it, specialised lymphoid tissues known * Correspondence and reprints.
E-mail address: [email protected] (G. Dougan).
as gut-associated lymphoid tissue (GALT) in the gut and the equivalent bronchial-associated lymphoid tissues (BALT) in the respiratory tract [18]. Pathogen-associated immune responses in the GALT and BALT drive potentially protective IgA production and local cellular responses. Immune responses at these sites have to have some capacity to discriminate between food/environmental antigens, which are non-threatening, and antigens from pathogens with the potential to cause damage and disease. The default mode at mature mucosal surfaces is to be non-responsive (in terms of the production of IgA and activation of effector mechanisms) and this factor limits immune responses to most antigens encountered at mucosal surfaces. However, it is essential that appropriate protective immunity, both innate and acquired, is rapidly stimulated against the pathogenic threat. At the moment, little is known about how the mucosal immune system regulates this balance. We do know that infection with many pathogens stimulates rapid mucosal and sometimes systemic responses that can lead to protection. We also know that certain products from microorganisms can activate immunity through interaction with specific receptors such as members of the Toll receptor family [22]. Toll receptors recognise generic molecules specific for microbes such as unmodified DNA, lipopolysaccharide and flagella. We also know that some microbial molecules that actively bind to
0923-2508/02/$ – see front matter 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 1 3 4 5 - 1
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S. Clare et al. / Research in Microbiology 153 (2002) 455–459
surface molecules on eucaryotic cells, such as toxins and fimbriae, are capable of acting as immunogens at mucosal surfaces [3]. With some antigens such as pertussis toxin or filamentous haemagglutinin from Bordetella pertussis, inactivation of cell binding activity has been shown to dramatically reduce mucosal but not systemic immunogenicity [6]. Thus, the mucosal immune system may be activated via targeted interactions involving pathogen products and immune cells.
2. Enterotoxins as mucosal immunogens and adjuvants Perhaps the best known examples of pathogen-derived mucosal immunogens are members of the cholera enterotoxin group typified by cholera toxin (CT) and the heat-labile enterotoxins (LT) of Escherichia coli. Microgram quantities of these toxins can stimulate potent mucosal and systemic antitoxin immune responses in a number of animals. Indeed, in some species these are so powerful that immune responses can also be stimulated to particular co-administered antigens that are not normally mucosal immunogens [28]. In other words CT and LT have potent mucosal adjuvant properties. Many studies on LT and CT have progressed in murine systems as these animals, unlike humans, have good tolerance to the toxicity of these molecules and immunisations can be conducted in the absence of significant side effects. Many murine studies have investigated the mucosal adjuvant activities of CT and LT using different bystander antigens and similar investigations are now being conducted in other species [35]. CT and LT are typical AB type toxins and share significant homology in terms of their primary and secondary structures. The A subunit of both toxins possesses ADPribosyltransferase activity that is responsible for the toxicity of the molecule. The A subunit is usually found tightly associated with the B subunit which is a pentemeric protein composed of five small identical polypeptides. The structure of both toxins have been solved using X-ray crystallography and they confirm that the toxins are very highly related [31]. The B subunit is the eucaryotic cell targeting domain and binds to non-protein receptors. CT has a high affinity for GM1 gangliosides, which are found in abundance on many eucaryotic cell types [16], whereas LTs are apparently more promiscuous in that in addition to GM1ganglioside they also bind to the glyco component of some glycoproteins [33]. After interaction of the B subunit with target cells the holotoxin enters endosomes and is transferred to the Golgi where the two subunits separate. The A subunit is directed in a retrograde manner through the Golgi where it eventually ends up in the cytosol. At this point the enzymatic activity of the toxin is expressed and this leads to the modification of host G proteins leading to the accumulation of cAMP in target cells. CT and LT can stimulate the production of secretory IgA, serum antibodies and potent cellular responses to co-
administered protein and in many infection models, protection, as has been demonstrated following CT or LT-based mucosal (oral or nasal) immunisation [17]. Significantly LT and CT can stimulate both CD4 and CD8-associated cellular responses [11,25]. The CD8 responses have been shown to be associated with cytotoxic activity mediating protection against viral infection [30]. Strenuous efforts have been made to decipher the mechanisms involved in the mucosal adjuvant activity of these proteins. The type of immune response associated with these toxins as adjuvants has been thoroughly described although the molecular basis of the activity remains elusive. Indeed, the toxins are likely to have pleiotrophic effects on both immune and non-immune eucaryotic cells complicating the interpretation of investigations [12]. A key question about the practical use of these toxins has been whether toxin activity can be separated from the adjuvant activities of the enterotoxins. Work with site-directed mutants of the toxin that inactivate ADP-ribosyltransferase activity but that do not significantly affect the stablility or assembly of the holotoxin has clearly shown that toxicity and adjuvant activity can be at least partially separated [10,11, 25]. LT mutants such as LTK63 (with an inactivating lysine amino acid substitution at position 63 of the A subunit) have now been well characterised in many different laboratories and they clearly retain adjuvant activity. It is also clear that the enzymatically active A domain also contributes, possibly in several different ways, to adjuvant activity [2,12].
3. Murine Citrobacter rodentium infection associated with mucosal immune disregulation Certain bacterial infection models also offer an opportunity to investigate the immune regulatory mechanisms operating during a live infection as well as the impact of immune disregulation on gut physiology. The murine pathogen C. rodentium possesses several features, which make it ideally suited to model both pathogenic E. coli infections and the immune mechanisms underpinning inflammatory bowel disease. Infection with C. rodentium is characterised by colonic hyperplasia primarily at the distal end of the colon and spreading up towards the caecum with increasing severity of infection [4]. The colon becomes swollen and more rigid as hyperplasia reaches a peak around day 14 postinfection. Wild-type animals resolve infection by day 28 and are resistant to rechallenge. The increase in colon size and weight can be attributed to cellular infiltration into the crypts and lamina propria as well as enterocyte proliferation. In severe infection the faeces become soft and in rare cases further complications, such as rectal prolapse, may ensue [19]. Immunostaining to localise bacteria reveals that in most wildtype infections C. rodentium is confined to the apical surfaces of the enterocytes. In immunocompromised mice and severe infections the pathogen appears to colonise deeper into the crypts as well as more extensively over the epithe-
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lial surface. This suggests host immune mechanisms play an important role in limiting infection to the mucosal epithelial surface and protecting deeper tissues. The mechanisms responsible for control are unknown and may depend upon a number of innate immune responses as well as physical factors such as mucus secretion and epithelial shedding. An important virulence-associated determinant of C. rodentium is the locus of enterocyte effacement (LEE), a 35-kb pathogenicity island with high homology to similar determinants encoded by both enterohaemorragic and enteropathogenic E. coli (EHEC and EPEC, respectively) [9, 21]. LEE-positive C. rodentium form characteristic pedestallike structures on the murine enterocyte surface known as attaching/effacing (A/E) lesions. The A/E lesions formed in mice are indistinguishable in morphology from those of EPEC on human enterocytes. A functional LEE is necessary and sufficient for bacteria to produce A/E lesions on epithelial cells [21]. Significantly, C. rodentium that lack LEE do not colonise the murine intestine to a significant level and do not induce colonic hyperplasia [27]. Like several other pathogenicity islands from intestinal pathogens, the LEE encodes a type three secretion system and associated effector proteins. The LEE-encoded apparatus allows secretion of bacterial effectors directly into host enterocytes to subvert normal epithelial (and possibly immune) function and allow intimate attachment of the bacterium to the host mucosa. Initial epithelial contact of C. rodentium with enterocytes leads to destruction (effacement) of the brush border microvilli. This is followed by translocation of the intimin receptor (Tir) and strong attachment of the bacterium via the intimin/Tir interaction. During this process the host cytoskeleton undergoes dramatic rearrangement leading to the formation of the characteristic pedestal of the A/E lesion. Experimental investigations of these critical pathogen/host mucosal interactions in vitro have been limited by the inability of simple cell culture to mimic the complex morphology and host reactions involved in the gut. The use of ex vivo explants from human biopsy samples and a murine model using C. rodentium have allowed substantial strides to be made both in molecular characterisation of the host-pathogen interaction and the immune mechanisms involved [29]. Attempts are now being made to understand the immune responses stimulated by C. rodentium infection and the mechanisms involved in protection and hyperplasia. Innate immune responses, inducing induction of antimicrobial peptides (defensins) can be detected during C. rodentium infection [29]. The localised production of nitric oxide by inducible nitric oxide synthase (INOS) can be clearly observed by immunohistochemistry, although it appears to play a minor role in immunity, since INOS-/-mice are no more susceptible to C. rodentium than wild-type [29]. This is distinct from a systemic infection such as Salmonella typhimurium where lack of INOS is almost always fatal [20]. Other innate immune mechanisms are less well studied in this model. For example the Toll-like receptors (TLRs) are likely to play
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an important role in macrophage, neutrophil and epithelial activation. The downregulation of TLR4 on epithelial cells, gut macrophages and T cells serves to limit the inappropriate response to commensal lipopolysaccharide [5,24] and this may be true of other molecules responding to bacterial components [32]. Whether the recruitment of immune cells to the lamina propria from systemic circulation leads to a breakdown in this tolerance and TLR activation is not known, but may be important for the strong Th1 response observed following C. rodentium infection [13–15,34]. Whilst TLR4 is unlikely to be the entire story mice lacking functional signalling from this molecule, e.g. C3HEJ, show increased susceptibility, higher colonic pathogen burden and greater hyperplasia when challenged with C. rodentium [4]. There has been much progress in recent years identifying the host immune mechanisms required for control. Many of the infiltrating cells are CD4+ and the cytokine milieu suggests a strongly polarised Th1 response [14,15]. Both interferon γ(IFN-γ) and interleukin 12 (IL-12) have been implicated in host defence against C. rodentium. IL-12−/− mice exhibit higher colonic and systemic bacterial counts and some succumb to systemic infections. Surviving animals take two weeks longer to clear infection than wild types. IFN-γ−/− animals also support higher pathogen loads and take approximately seven days longer to clear infection than wild-type [29]. The role of TNF-α in Crohn’s disease and other forms of inflammatory bowel disease is well documented and antiTNF-α therapy is often effective in ameliorating symptoms [1,7,26]. Whilst in C. rodentium infections TNF-α is not required for clearance, mice lacking TNF-α receptor show higher bacterial burdens, increased IL-12 production and greater hyperplasia, but resolve infection as rapidly as wild types [13]. The fact that lack of the TNF-α receptor induces greater hyperplasia suggests that TNF-α may modulate the Th1 mucosal response and serve to limit pathology in wild-type animals. Studies in RAG1 knockout mice (lacking a T- and B-cell repertoire) have revealed that these cells are required for elimination of the infection. These mice fail to clear the bacteria and often succumb to infection [34]. CD4+ -cell-depleted mice fail to clear infection and antiC. rodentium serum antibody titres are significantly reduced compared to nondepleted animals (Simmons et al., in press). Depletion of CD8+ cells has no significant effect upon clearance or pathology. The role of B cells has been further clarified by the use of Igh6−/− mice (B-cell-deficient) (Simmons et al., in press). These animals remain hyperplastic and fail to resolve infection until at least day 56 postinfection, although they do not succumb to systemic disease. Thus it would appear that antibody production is required for clearance of C. rodentium and CD4+ T-cell help is needed to produce a resolving level of antibody.
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4. Conclusions As we learn more about the molecular mechanisms underpinning the pathogenicity and immunology of mucosal bacterial infections new opportunities are emerging to generate novel therapeutic approaches. Bacteria activate the mucosal and systemic immune systems through specific cues that allow immune cells to identify their presence. The location of bacterial antigens within the context of the mucosal surfaces may be critical and the ability of products to interact actively with immune cells may stimulate active immune responses. Bacterial products such as enterotoxins will continue to be used as useful probes of the mucosal immune system and further studies on pathogens such as C. rodentium, that induce immune pathology at mucosal surfaces, will be used to model conditions such as inflammatory bowel disease and provide routes towards therapies.
Acknowledgements This work is supported by The Wellcome Trust.
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