Mucosal Immune Responses to <b><i>Escherichia coli</i></b> and <b><i>Salmonella</i></b> Infections
ODILIA L. C. WIJBURG AND RICHARD A. STRUGNELL*
[SECTION EDITORS: FERRIC FANG AND MARTIN KAGNOFF]
Posted June 6, 2006
Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3010, Australia
*Corresponding author. Phone: 61-3-8344 9919, Fax: 61-3-9347 1540, E-mail:
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The mucosal epithelia of the respiratory, urinary, and gastrointestinal tracts as well as the ocular conjunctiva and the inner ear are continuously exposed to potentially pathogenic microorganisms and other harmful substances as well as commensal microbiota and harmless food antigens. A variety of immune and nonimmune mechanisms cooperate to protect the mucosal surfaces from invading pathogens. The highly specialized mucosa-associated immune system has evolved to discriminate between innocuous and harmful antigens, and it functions in cooperation with nonimmune mechanisms to eliminate pathogens while maintaining a tolerogenic state toward harmless antigens. Nonimmune defense mechanisms include physical and mechanical barriers, such as the acidic environment of the stomach, secreted bile salts and proteases, tight junctions between epithelial cells, gastrointestinal peristalsis, and motility mediated by ciliated epithelial cells. Mucus, produced by goblet cells, is a viscous substance composed of glycoproteins that form a film over the gastrointestinal epithelium that is constantly moved along by ciliary action. Mucins may protect the mucosal epithelial surface by preventing the attachment of microorganisms and/or toxins to epithelial cells, and by binding secretory immunoglobin A (SIgA) mucins may prevent contact of antigens with the mucosal surface. The antibacterial substances lactoferrin, lysozyme, and peroxidase are present in most external secretions (including mucus) and have been shown to inhibit bacterial growth by reducing uptake of iron by bacteria, by damaging the bacterial cell wall resulting in lysis of the cells, and through the generation of superoxide radicals, respectively. The resident microflora of mucosal surfaces forms another important innate defense mechanism; their presence forces invading bacteria to compete for attachment and colonization sites and essential nutrients.
The concept of a specialized, adaptive mucosa-associated immune system was first developed by Alexandre Besredka, who in 1919 at the Institut Pasteur in Paris demonstrated that local immunity in the gastrointestinal tract (GIT) or skin could be induced independently of systemic immunity during experimental oral infections with enteric bacteria or skin infections with Bacillus anthracis and that this was of great importance for the host’s resistance to gastrointestinal or cutaneous pathogens (11). Studies in the 1940s by Burrows, who studied an experimental cholera toxin vaccine in guinea pigs, showed a correlation between the presence of fecal antibodies (coproantibodies), but not serum antibodies, and protection against oral infection (21, 22). The theory of the existence of a local, secretory immune system developed further with the discovery of the IgA class of antibodies by Heremans et al. in 1959 (61). The concept was bolstered with the observation that many different external secretions that cover mucosal surfaces, as well as breast milk, contain predominantly IgA (26, 57) and the demonstration of a predominance of IgA-containing plasma cells in the parotid gland and the small intestine (27). Since then, the presence of IgA has been considered one of the hallmarks of the mucosal immune system, and it has been shown that secretory IgA is a polymeric molecule (mainly dimeric) in which the IgA molecules are joined by a unique polypeptide or J-chain, which specifically binds to the polymeric Ig receptor (pIgR) expressed on the basolateral surface of epithelial cells (17). The pIgR/dIgA/J-chain complex is transported to the apical side of the epithelial layer, where the complex is proteolytically cleaved and SIgA is released into the mucosal lumen. The ectoplasmic domain of the pIgR, known as secretory component (SC), remains associated with the SIgA (Fig. 1) (76, 178). The SC may protect SIgA from proteolysis and may also target the SIgA to mucus by selectively binding to mucins (140). A recent comprehensive review on regulation of intestinal IgA synthesis has been provided (44). Protection of the host by SIgA is thought to occur by three mechanisms (Fig. 1). First, SIgA may prevent adhesion of bacteria to the epithelial cells, thereby preventing colonization and invasion. Second, SIgA may neutralize pathogens intracellularly (20). Finally, SIgA may be involved in antigen exclusion, whereby antigens are bound to dimeric IgA molecules in the lamina propria and transported into the lumen after binding to the pIgR and transcytosis (149).
The best-characterized mucosa-associated lymphoid tissue (MALT), and also the most relevant for this chapter, is the gastrointestinal-associated lymphoid tissue (GALT) (Fig. 2). In general, the GALT is compartmentalized into inductive and effector sites (reviewed in detail in references 120, 123). The organized lymphoid tissues of the GALT that are involved in induction of immune responses are the Peyer’s patches (PPs), the mesenteric lymph nodes (MLNs), and the isolated lymphoid follicles (ILFs) that are scattered throughout the lamina propria of the small and large intestines. The epithelium that covers the PPs and ILFs is referred to as follicle-associated epithelium (FAE) and lacks the brush border typical of normal absorptive epithelium. The FAE, and the area immediately underneath the epithelium known as the subepithelial dome (SED), contain many lymphocytic cells as well as macrophages and dendritic cells. The most characteristic feature of the FAE, however, is the presence of M cells (microfold cells), a highly specialized cell type that is responsible for the controlled uptake and transport of antigens from the mucosal lumen to the subepithelial lymphoid tissues. Typically, M cells differentiate from enterocytes, but they lack microvilli and are not covered by a mucoid glycocalyx. M cells do not express major histocompatibility complex class II (MHC-II) molecules, and are therefore unable to present antigens directly to T lymphocytes present in the lamina propria. In contrast, epithelial cells, which may also transfer absorbed antigens to the mucosa, are able to present antigens to CD4+ T lymphocytes. A third mechanism by which antigens may gain access to the inductive lymphoid tissues is via direct sampling of the intestinal lumen by epithelial dendritic cells (147). These dendritic cells express tight junctions and may protrude their cellular processes into the lumen without disturbing the integrity of the epithelial barrier.
Multiple subsets of dendritic cells that may function as antigen-presenting cells (APC) have been identified in the PP and the SED and the lamina propria of the small intestine, including the myeloid CD8α–CD11b+ dendritic cells and the lymphoid CD8α+CD11b− dendritic cells, as well as an "unusual" CD8α–CD11b– subset (73, 74, 75). Dogma states that dendritic cells in the SED and PP take up, process, and present the antigens sampled by the M cells to T lymphocytes in the PPs, and that these activated T lymphocytes then migrate via the lymphatics, MLNs, and bloodstream, to the mucosal surface. However, the identification of multiple dendritic cell types in the lamina propria and among the epithelial cells, and the observation that these dendritic cells may sample the gut lumen directly, or may take up luminal antigens transported by epithelial cells, together with the observed constitutive trafficking of dendritic cells from the lamina propria to the MLNs, have led to the hypothesis that lamina propria dendritic cells may, rather than interact with T lymphocytes locally, migrate directly to the MLN where they activate antigen-specific T lymphocytes (103). Dendritic cells in the PPs respond differently to stimulation than dendritic cells in the spleen, and may react by producing interleukin 10 (IL-10) instead of IL-12 and stimulate T lymphocytes to produce Th2 cytokines (73, 75, 194). It has been suggested that this altered response reflects an ability of some specific subsets of dendritic cells in the MALT to induce tolerance (120).
The main effector site of the GALT is the lamina propria outside of the PPs and ILF. Lymphocytes primed in the PP or MLN selectively upregulate expression of α4β7 integrin, and following migration through the thoracic duct and bloodstream, are redirected back to the gastrointestinal mucosa by interaction with the mucosal addressin cell-adhesion molecule a (MAdCAM1), the ligand for α4β7 that is highly expressed on vascular endothelial cells in the GALT and in the mammary glands (9). In addition, chemokines may be involved in the specific migration of mucosally primed lymphocytes to the mucosa; at least in mice, CCL25 has been shown to specifically attract IgA+ B cells (16). These B lymphocyte blasts are activated in the PP follicles and switch from producing IgM to producing IgA, and further mature into IgA-producing plasma cells in the lamina propria. As described before, the constitutive secretion of large amounts of dimeric IgA, up to 40 mg/kg in the normal human gut, is one of the hallmarks of the mucosal immune system. Dimeric IgA, as well as pentameric IgM, is secreted into the mucosal lumen via the pIgR (76, 178), and in general, it is assumed that these secretory antibodies form a first layer of defense against potential pathogens.
It has recently been shown that, following antigen-specific stimulation, most effector memory T lymphocytes accumulate preferentially in the lymphoid tissues, in particular, in the lamina propria (109, 146). These CD4+ and CD8+ lymphocytes, besides α4β7, all express CCR9, the ligand for chemokine CCL25 that is expressed in the thymus and in the small intestine of mice and humans (89, 134). The CD4+ T lymphocytes mainly reside in the lamina propria, are unresponsive to stimulation through their T-cell receptor (TcR), and secrete large amounts of cytokines, in particular, interferon gamma (IFN-γ), IL4, and IL10, which may help B-lymphocyte blasts to produce IgA. It has also been suggested that these CD4+ T lymphocytes are of particular importance in the maintenance of tolerance and function as regulatory T cells (85).
A majority (60%) of the CD8+ T lymphocytes reside between the epithelial cells (hence their designation as intraepithelial lymphocytes [IELs]) and possess cytotoxic abilities. A large proportion of IELs express the CD8αα homodimer instead of the CD8αβ heterodimer that is expressed by most other CD8+ T lymphocytes. In addition, the CD8αα T lymphocytes may express the γδTcR instead of the αβTcR (91). It is well known that the CD8αα+ T lymphocytes originate from an extrathymic site, but their precise ontogeny remains debated (58). Some investigators have suggested that the cells originate from progenitors in cryptopatches, which are postnatal lymphoid aggregates present among the crypts of the intestinal wall (168). Specific activation of IELs has been demonstrated following infection of the host with pathogens, but no specific role in the defense of the GIT has been attributed to CD8αα T lymphocytes.
Escherichia coli and the Salmonella spp. typically infect the host through the gastrointestinal mucosa. In this process, these bacteria induce many different host responses, including elements of both the innate and acquired immune systems. It has long been held that these responses are important for immunity to infection—the recent development of powerful tools, principally in the form of genetically manipulated mice that allow the mucosal immune response to be more fully analyzed, has challenged some central beliefs with respect to the role of the mucosal immune system. This chapter reviews our understanding of the importance of mucosal immune responses in resisting infections caused by E. coli and Salmonella spp.
The species E. coli is a member of the normal flora in many animal species and is responsible for an array of serious systemic and localized infections, including septicemia, meningitis, cystitis, gastroenteritis, and hemolytic-uremic syndrome (82). The pathogenesis of various E. coli infections will be covered elsewhere in EcoSal (see Domain 8.5)—the effective immune response necessary to prevent or limit serious disease from the diverse infections caused by E. coli will almost certainly depend on the distinct pathological mechanisms. For example, the SIgA antibodies that are necessary to limit pathology by gut toxins liberated by enterotoxigenic E. coli are of a different isotype from the opsonic antibodies required for phagocytosis of encapsulated, septicemia-associated E. coli. This section focuses on the major human E. coli infections and discusses whether antigen-specific mucosal immune responses are important for resistance against primary infection or reinfection by pathogenic E. coli.
Pathogenic intestinal E. coli are currently divided into six categories: enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and diffusely adherent (DAEC) E. coli. The extraintestinal E. coli include uropathogenic (UPEC) and meningitis-associated (MNEC) E. coli (see Domain 8.6 in EcoSal) (82, 148). Each of these pathotypes expresses an array of virulence determinants that are important in pathogenesis—fimbriae for adhesion, secreted proteins as mediators of local and systemic pathology, and Toll-like receptor (TLR) ligands including lipoproteins, lipopolysaccharide (LPS), flagella, and DNA (59, 122) (Table 1 and 2).
Table 1E. colipathotypes and typical lesions |
Table 2Ligands for toll-like receptor ligands in Enterobacteriaceae |
Three basic types of lesions can be induced by gastrointestinal E. coli: (i) apical surface adherence followed by secretion and cellular uptake of bacterial exotoxins, (ii) attaching-effacing (A/E) lesions mediated by bacterial type III secretion system (TTSS) effector proteins, and (iii) epithelial cell invasion (Fig. 3); these pathological processes are covered in more detail elsewhere in EcoSal (see Domain 8.5). The following sections will analyze human data on mucosal immunity against E. coli, a growing body of data of mucosal responses in food-production animals and other natural hosts of E. coli, and more recent experimental studies in mice carrying defined deletions in genes encoding specific immunological effectors, to show that there may be considerable conservation of the effective host mucosal immune response against this pathogen.
It might be hoped that, after 150 years of infectious disease research, our understanding of bacterial pathogenesis and immunity would be such that we could clearly define the type and specificity of the immune response necessary to prevent or cure most important human bacterial infections. Unfortunately, this is not the case. Although there have been some notable successes (e.g., antibody specific for exotoxins such as tetanus toxin) and capsular polysaccharides (e.g., Haemophilus influenzae capsule), the immune correlate of protection, the in vitro measurable immune parameter(s) corresponding to protection against a specific infectious disease, is poorly understood for many E. coli infections.
In general, it has been difficult to define widely accepted correlates of protection against human gastrointestinal infections because (i) potential effectors (e.g., SIgA or lymphocytes trafficking to the gut) are difficult to obtain and transfer to infection-naïve recipients, and (ii) defined vaccines that are fully protective against many types of E. coli infection have yet to be developed. Modern vaccine development typically facilitates the identification of correlates of protection, whether specific antibodies, T cells, or nonspecific effectors such as macrophages. Where human ETEC vaccines have been developed for the prevention of traveler’s diarrhea, the lack of effective vaccines against human EPEC infections, arguably the most important E. coli causing human disease, has meant that much of our understanding of immunity in EPEC infections has been extrapolated from studies of related bacteria in animal models. Rabbits infected with rabbit enteropathogenic E. coli (REPEC) and mice infected with Citrobacter rodentium have provided some key insights into the capacity of immune responses to limit both primary infection and reinfection by bacteria that induce A/E lesions.
A direct cause-and-effect relationship between SIgA and immunologically mediated protection is especially difficult to establish in human subjects. Unlike serum antibody isotypes, which can be passively transferred to naïve individuals to determine their protective potential, secretory antibodies exhibit a very short half-life in serum. Passive transfer of "secretable" IgA antibodies in serum results in their rapid secretion through all mucosal epithelia that express the pIgR on the basolateral surface of the epithelium, the basis of the so-called "common mucosal immune system." The concentrations of SIgA that might be found locally in the gut from local B-cell activation cannot therefore be obtained by passive transfer of antibodies alone.
In vitro correlates of immunity generated in studies of animal models, natural infections in humans, and from participants in vaccine trials are typically supported by analysis of human subjects who carry genetic immunodeficiencies or who are more specifically immunocompromised by co-morbidities or infections such as human immunodeficiency virus (HIV)/AIDS. IgA deficiency is one of the most common immunodeficiencies, providing a sizeable cohort for studying the role of IgA in disease prevention, although compensation by secretory IgM may ameliorate some of the clinical consequences of sIgA deficiency per se (see, e.g., reference 143). Human IgA deficiency is associated with an increase in minor respiratory tract infections, typically viral, and a limited study of E. coli carriage in healthy and immunodeficient individuals suggested that the E. coli carried by IgA-deficient individuals express more putative virulence determinants than those carried by healthy individuals (49). It is assumed from the analysis of these studies that the absence of IgA in the secretions leads to selection of E. coli more commonly associated with disease. No attempt was made to quantify the total number of E. coli present so it is not known whether the absence of IgA antibodies increases total colonization by the bacteria as members of the normal flora. An increase in bacterial infections of the GIT is not a noted feature of IgA deficiency although recalcitrant giardiasis is sometimes observed, suggesting that control of this eukaryotic gut parasite is IgA dependent.
Since most E. coli infections occur in the developing world, there has been only limited systematic investigation of co-morbidities or associated infections; however, in contrast to infection with Salmonella enterica serovar Typhimurium, HIV/AIDS is not seen as a major risk factor for gastrointestinal E. coli infections, despite an increase in rates of diarrhea in people infected with HIV (77). This observation would suggest that CD4+ T cells are less important than other immune effectors in resistance to E. coli infections. Some additional information concerning protective immunity to E. coli has been obtained from cognate (i.e., nonmodel) E. coli infections in food production and other animals including cattle, pigs, and rabbits. Cattle infected with E. coli are the direct source of disease in some food-borne EHEC outbreaks (see Domain 8.0 in EcoSal), and the development of veterinary vaccines for use in food production animals (e.g., to prevent E. coli diarrhea in piglets known as "porcine scours") has increased our understanding of immunity in E. coli infections in humans. What is clear from studies in humans and veterinary species is that immunity against reinfection with E. coli of the various pathotypes can be induced and that much of this immunity depends on virulence-determinant-specific antibodies.
One issue central to effective mucosal immunity against E. coli adhesins and toxins is the importance of eliciting secretory antibody responses. It is widely held that SIgA specific for secreted proteins of E. coli is fundamental to immunity on the apical epithelial surface, a position supported, until recently, by observation, correlation, and extrapolation, rather than by direct experimentation. With the development of genetically modified mice that lack key components of the secretory immune system (e.g., J chain, pIgR, and IgA), it is now possible to directly test the role of secretory antibodies in protection against infection in murine models of disease. As we shall see, these studies directly support a role for secretory antibodies in the inhibition of toxicity but have yet to reveal a major role for secretory antibodies in blocking in vivo adhesion of many pathogens. The most compelling evidence that antibodies per se can block E. coli colonization has come from studies in sows immunized against fimbriae, which passively transfer this antibody in their milk to suckling piglets, to provide protection against ETEC colonization and disease (119, 124).
Immunity against E. coli Fimbrial Adhesions.
Molecular analysis and dissection of E. coli fimbriae has generated much of what we know of fimbria or pilus biogenesis (69, 70). In some cases, the receptor for the E. coli fimbrial tip adhesin subunit has also been identified (96). Analysis of receptor distribution usually lends weight to the argument that the distribution of a major fimbrial receptor, in part, determines the host and tissue tropism of pathogens like E. coli and underscores the pivotal role that initial fimbria-mediated adhesion plays in the pathogenic process for many gram-negative bacteria (8), an observation reinforced by numerous fimbrial gene mutagenesis studies.
The impact of ETEC infections on Western tourists has meant that the ETEC are the best-studied E. coli infections in their natural (i.e., human) host. The incidence of ETEC disease in Western travelers has stimulated the detailed analysis of virulence determinants including the fimbriae, LPS, flagella, and toxins of ETEC from many sources, and the development of ETEC vaccines, which have recently been the subject of large-scale immunogenicity trials (154, 155, 156), provides a near-term opportunity for the prospective analysis of mucosal immune responses in protection. ETEC vaccines typically comprise ETEC fimbrial antigens and/or modified nontoxic enterotoxins and have demonstrated moderate to good immunogenicity in clinical trials—the end point of ETEC vaccine trials is still the subject of some discussion, and at present there are only limited published protection data linking anti-fimbrial immune responses with vaccine efficacy.
The best-characterized E. coli fimbrial adhesins with respect to mucosal immunity are colonization factor antigens (CFAs) of ETEC; related structures are called E. coli surface antigens (CSs) or putative colonization factors (PCFs) (41, 129, 195) (see elsewhere in EcoSal). The CFAs are thin, typically plasmid-encoded fimbrial adhesins. CFA/I was the first CFA to be characterized, and a very large family of sequence-related and unrelated CFAs have subsequently been identified. CFAs facilitate attachment of ETEC to small intestine epithelial cells and hence initiate the colonization events that lead to toxemia.
The antigenic complexity of the ETEC fimbriae has required that ETEC fimbria-based vaccines contain multiple fimbrial components—the most recent vaccine included CFA/I, CS1, CS2, CS3, CS4, and CS5 (155). Studies of these vaccines in humans have commonly attempted to elicit CFA-specific SIgA through oral immunization, achieved in part by formulating the complex vaccine with the recombinant cholera toxin B-subunit (rCT-B) as a mucosal adjuvant. Epidemiological analyses of human responses to ETEC have suggested that exposure to ETEC correlates with the production of fimbria-specific SIgA (167), and it is inferred from these studies that the SIgA, induced by infection or immunization, can provide resistance against reinfection, since serotype-specific immunity has been noted in very limited studies in humans (41, 129, 195). This concept is supported by studies in animal models (2).
Purified ETEC CFAs were used as vaccine antigens in a limited study in humans (42)—oral administration of CFAs following parenteral vaccine priming induced secretory antibody responses that appeared to be protective in a very small vaccinated cohort. In short, it is not yet known whether immunity against fimbrial adhesins will be protective against human ETEC disease under field conditions; certainly, the antigenic diversity among the ETEC fimbriae present some serious vaccine formulation challenges. However, the most recent analysis of immunogenicity of the hexavalent fimbrial vaccine, combined with CT-B, provides some hope since this preparation was both well tolerated in young Bangladeshi children and immunogenic (145).
The most convincing data on the potential of antibodies to block E. coli infections have been provided from studies in pigs. The counterparts to the human ETEC CFAs in pig ETEC are the fimbrial adhesins K88, K99, F4, and 987P. These fimbriae are used as vaccine immunogens in pigs in a commercial vaccine, Porcine Pili Shield™ (Novartis Animal Health). This vaccine, which is effective in preventing ETEC infections in weanling piglets, is administered parenterally to sows, which then transfer protection in milk to suckling piglets. While the isotype of the antibodies providing protection is not known (some analyses suggest a correlation between levels of specific IgA present in the milk and protection), multiple studies have demonstrated that the maternal transfer of fimbria-specific antibodies limits both colonization by porcine ETEC and shedding of bacteria expressing the same fimbrial antigens (see chapter Vaccines against Infections Caused by Salmonella, Shigella, and Pathogenic Escherichia coli for additional details).
The role of antifimbrial antibodies in the prevention of non-GIT (i.e., UPEC) infections has been studied in animal models, including nonhuman primates (90). A vaccine based on FimH, the terminal adhesin of the Pap pili, provided protection against bladder infection when administered parenterally and generated an IgG antibody response. This suggests that IgG antibodies are sufficient for protection against fimbria-mediated adhesion of E. coli to the bladder epithelium of primates.
Is SIgA essential for immunity to fimbria-mediated adhesion in the gut? The fact that parenteral immunization with fimbriae or terminal fimbrial adhesins can provide both passive and direct protection against bacterial adhesion would argue against this, because injected vaccines do not normally induce significant levels of SIgA. Studies that demonstrate the ability of specific serum antibodies to inhibit fimbrial binding suggest that pIgR-mediated transport of antibodies may not be essential to block adhesion. How might serum antibodies reach the apical surface of the epithelium? Serum antibodies could be exported to the apical surface paracellularly under inflammatory conditions, in which the permeability of epithelial tight junctions is increased, or transcytosed through specific receptors such as FcRn on epithelial cells (33). For example, transudated serum antibodies specific for type 1 fimbriae, which are commonly expressed by a range of enteric flora, could provide protection against related pathogens, including Salmonella spp. and E. coli, once focal infection and inflammation are initiated. The importance of SIgA in protection has significant consequences for immunization protocols since parenteral immunization does not typically induce SIgA; neutralizing serum antibodies are much easier to achieve with existing adjuvants and immunization protocols. Experience with other bacterial vaccines argues both for (Vibrio cholerae Dukoral™) and against (Bordetella pertussis acellular vaccine) the necessity for mucosal delivery to achieve pilus/fimbria-mediated immunity to infection by mucosal pathogens.
In summary, there is good evidence that fimbria-specific antibodies, on reaching the gut or urinary tract lumen, can block colonization by E. coli and/or shedding of the bacterium. Whether these antibodies must be actively transported (i.e., belong to the IgA or IgM isotypes carrying J-chain), or whether transudation of serum antibodies through the epithelium via paracellular leakage following focal inflammation is sufficient to control colonization, is yet to be determined.
Immunity against E. coli Exotoxins.
The enterically active exotoxins secreted by E. coli are responsible for very severe and sometimes lethal symptoms in diseases caused by a variety of E. coli pathotypes including the ETEC, the EHEC, and EAEC (82, 148).
In contrast to the somewhat ambiguous data surrounding the absolute requirement for SIgA in the neutralization of fimbrial adhesion, there is direct evidence that (i) SIgA can block the epithelial uptake of the ETEC heat-labile toxin (LT), and (ii) LT binding and uptake are inhibited by pIgR-secreted SIgA and not by transudated serum IgG (see Fig. 4). The LT exotoxin family contains a number of seemingly minor sequence variants (165) (see elsewhere in EcoSal) and is one of a small number of proteins that are potent mucosal immunogens (29), that is, immunogens that can avoid the degradative processes of the GIT and elicit an immune response from the lumenal side of the epithelium. LT is a classical AB toxin—the B subunit (LT-B) is the binding domain and the A subunit (LT-A) is an enzyme with two activities, NAD glycohydrolase and ADP-ribosyltransferase (165). LT binds to the cell surface receptors including the ganglioside GM1, is internalized, and triggers an increase in cAMP, which in turns inhibits an ion channel, leading to an osmotic diarrhea (see Domain 8.7 of EcoSal). Murine models of LT toxemia have been developed, taking advantage of a growing number of mouse strains lacking specific components of the immune system. These models have used nontoxic but immunogenic doses of LT (or the closely related cholera toxin [CT]) to stimulate strong mucosal humoral responses prior to challenge with a toxic dose of enterotoxin. After dissection of the gut, the weight of the gut tissues as a proportion of the animal’s body weight can be calculated (Fig. 4). Analysis of CT challenge models in mice lacking the J chain or the pIgR (Fig. 4) have revealed that mice without an intact SIgA secretory system are unable to block the osmotic effects of CT (101) despite high serum levels of CT-specific serum IgA and IgG. Assuming that E. coli actively secrete LT, as demonstrated by Tauschek et al. (174), then it is very likely that SIgA specific for LT will protect against LT-mediated ETEC disease in humans.
The close antigenic relationship between LT and CT (the proteins are approximately 80% identical at the amino acid level) has been exploited by vaccine developers, and there is an oral cholera vaccine (Dukoral™, Berna) that has shown significant (ca. 50 to 65%) short-term protection against ETEC infections (157).
The other important family of ETEC toxins, the heat-stable toxin (STs), present challenges different from LT in that they exhibit very poor native immunogenicity. The STs are small, tightly folded (2-kDa) single-peptide, protein toxins that bind to guanylyl cyclase C, causing an increase in cGMP and, ultimately, excessive fluid secretion from the epithelium (125) (see elsewhere in EcoSal). The poor immunogenicity of ST has been addressed by fusing the protein or portions of the protein, genetically or chemically, to carrier proteins including LT-B and flagellin (48, 86, 138). These fusions have increased ST immunogenicity, with the antibodies raised conferring some protection against the osmotic effects of ST, but the fusion proteins have yet to be fully tested in humans. Studies of diarrhea in developing countries have revealed that ST-producing ETEC are a major cause of diarrhea, especially in the young. In a prospective study of rotavirus in Venezuela, ETEC-expressing STs were isolated from 30% of cases of pediatric diarrhea, compared with 6% with rotavirus and 10% with LT-producing ETEC (139). Clearly, there is a pressing need to develop a mucosally active vaccine against ST.
Another family of medically important mucosally active exotoxins produced by EHEC is the Shiga toxins (Stx) or verocytotoxins (VTs) chapter (Shiga Toxins: Potent Poisons, Pathogenicity Determinants, and Pharmacological Agents). Stx are a family of AB toxin that binds to the glycolipid Gb3; the A subunit cleaves ribosomal RNA, inhibiting protein synthesis and inducing "ribotoxic stress" (164, 175). The toxins are produced in the large intestine, can cause local epithelial damage through apoptosis of epithelial cells, manifested by bloody diarrhea, and also travel through the epithelium via the bloodstream to the kidneys, where they cause renal injury. The immunogenicity of Stx is poor relative to LT and, in mice, the level of response elicited by Stx-B is related to the H-2 MHC haplotype of the animal (6). The attention attracted by large, recent outbreaks of EHEC infections in humans in developed countries has stimulated the development of Stx vaccines based on mutant, detoxified toxins and adjuvant formulated Stx-B. Some of these vaccines have generated immune responses that appear to be effective in blocking epithelial toxicity and the systemic effects of Stx toxemia in experimental challenge systems including primates, rabbits, and mice (72, 98, 106, 191). They are yet to be formally tested in humans.
Pigs develop a serious disease called porcine edema disease (ED) cause by non-EHEC serotypes of E. coli (i.e., O138, O139, O141) carrying the Shiga toxin Stx2e (121). This natural disease serves as a model for EHEC infections in humans and reveals how vaccines might be used to prevent EHEC infections in humans. Some protection against ED was observed in pigs given parenteral vaccines comprising formalin-inactivated Stx2e, which was increased when genetically detoxified Stx was used in the vaccines (15). Fimbriae from ED strains, given orally, have also provided protection against ED pathology. Protection against death from ED in pigs has been seen in animals given a live oral vaccine containing E. coli expressing a mutant Stx carrying amino acid substitutions in the Stx-A subunit (104, 131). The enhanced protection of pigs observed following oral vaccination with the live bacterium expressing an attenuated Stx, or with bacteria carrying the ED ETEC fimbriae (10), would suggest that resistance at the mucosal surface might be conferred against serious EHEC disease through nonparenteral immunization.
Other GI and non-GI pathotypes of E. coli secrete a variety of enterotoxins that have not yet been the subject of vaccine studies; these include the Shigella enterotoxin 1 of EAEC. It is therefore not yet clear whether mucosal immune responses are fundamental for protection against these toxins. The non-GIT UPEC pathogens secrete a hemolysin (HlyA), a type I-secreted toxin, RTX (RTX stands for "repeats in the structural toxin"), which shows sequence similarity to an RTX toxin from EHEC (7) and a vacuolating protease (Sat) (82). Patients with UPEC urinary tract infections develop antibodies that neutralize the lytic activity of HlyA in vitro (158). These neutralizing antibodies are of an IgG class, and it is not known whether they are functional in vivo. While some studies have attempted to resolve the antigenic complexity of the HlyA family, no vaccine trials have been conducted to date, so it is not clear whether antihemolysin serum antibodies are sufficient to inhibit HlyA-mediated pathology. The conservation of HlyA sequences among UPEC has been used to support the argument that a broadly cross-reactive HlyA-based vaccine might be feasible (130).
A number of human studies have attempted to resolve whether immunity against reinfection with EPEC, the major group of A/E E. coli, exists, and whether this immunity is correlated with the appearance of antibodies in serum or colostrum. Endemic transmission of EPEC infections occurs predominantly in children less than 12 months of age, suggesting that older children are immune to the low levels of bacteria that circulate outside of "common source" outbreaks. Donnenberg et al. investigated the capacity of adult volunteers to resist reinfection and observed that, while prior exposure did not seem to stop the disease from occurring, previous infection with EPEC reduced the symptoms associated with virulent challenge (35).
The capacity of mothers to transfer protection against EPEC diarrhea to newborns through colostrum and milk has been extensively analyzed, and protection against EPEC disease appears to correlate with IgA specific for the major virulence determinants of EPEC including the bundle-forming pilus (Bfp) adhesin chapter (Adhesins of Enteropathogenic Escherichia coli), the A/E type III secretion apparatus chapter (Adhesins of Enteropathogenic Escherichia coli), Intimin chapter (Adhesins of Enteropathogenic Escherichia coli) and its cognate receptor Tir chapter (Adhesins of Enteropathogenic Escherichia coli), and LPS (e.g.. 24, 31, 32). Breast feeding per se is known to be of benefit in preventing EPEC disease—taken together, these data would indicate that maternally transferred IgA is capable of limiting colonization and/or pathology associated with EPEC. The common conundrum concerning the essentiality of SIgA in protection remains, however, since children recovering from EPEC infection develop EPEC virulence determinant-specific serum (i.e., IgG) antibodies.
The absence of suitable murine models of ETEC and EPEC infection has limited our understanding of the specific role of mucosal effectors in preventing E. coli infections and led to the use of related pathogens as surrogates. Citrobacter rodentium is a murine pathogen that carries a locus of enterocyte effacement similar to that in EPEC and EHEC and induces A/E lesions in the murine large bowel that mimic those seen in human EPEC and EHEC infections (100). Infection of mice carrying defined immunodeficiencies in T cells, B cells, and the secretory response has revealed that a CD4+ T-cell-dependent antibody response is essential for the resolution of A/E lesions caused by C. rodentium (18, 102, 162). This antibody response is independent of antibody secretion, insofar as mice lacking the pIgR, J chain, or IgA showed normal clearance of the bacterium. Mice lacking B cells (μMT) or CD4+ T cells developed very serious or fatal disease, and protection was achieved by passive transfer of IgG antibodies specific for C. rodentium. These data support a major role for high-affinity, antigen-specific serum IgG antibodies in the resolution of A/E lesions. Can these results be extrapolated to infections in humans? The requirement for CD4+ in clearing C. rodentium infections would argue that HIV might be an important risk factor in EPEC and EHEC infections. However, studies to date have revealed that, while monkeys and especially infant monkeys appear to be at significant risk from EPEC infections, people with HIV/AIDS do not appear to suffer more serious E. coli infections, with the exception of one report of rare events (12). While this lack of association could be explained by a failure to distinguish commensal E. coli from E. coli pathotypes, a better argument might be that the profound immunodeficiency associated with HIV/AIDS does not manifest until after the period in which infants in the developing world are most at risk from EPEC, i.e., the first 12 months of life. The development of protective EPEC-specific antibodies during this period may subsequently afford protection as CD4+ T-cell counts decrease with age.
Studies in rabbits infected with EPEC (i.e., REPEC) have shown that the diarrheal phenotype of these bacteria depends on the expression of fimbriae (e.g., Ral [1]), which are thought to mediate initial or so-called distal adhesion allowing the type III secretion products of the locus of enterocyte effacement to drive intimate adhesion. Fimbrial adhesion may also be important in EPEC infections in humans where in vitro studies of the Bfp suggest that the aggregation and adhesion mediated by Bfp are probably essential to EPEC pathogenesis. It is not clear whether immunization against fimbrial adhesins provides solid protection against EPEC or REPEC infections in their natural hosts and whether disease resistance will depend on SIgA.
The observation in mice that serum antibodies offer protection against A/E E. coli is supported by studies in cattle immunized against A/E EHEC (144). Cattle immunized with a parenteral vaccine containing EHEC secreted proteins, including the A/E type III secretion apparatus and secreted proteins EspB, EspD, and Tir, appeared to shed fewer bacteria following challenge. A vaccine prepared from a strain that did not express Tir was less effective, suggesting that Tir might be an important target for the systemic immune response elicited by the vaccine.
The pathogenesis of the EIEC appears to be dominated by the type III secretion system encoded on the EIEC virulence plasmid (82). The EIEC are taxonomically very similar to Shigella spp., which, unlike the EAEC, have been the subject of a number of vaccine programs. Several different types of Shigella vaccines have been developed and tested in humans, including live attenuated vaccines and adjuvanted subunits (88, 137); to date, protection seems to be enhanced following oral delivery, although recent parenteral vaccines based on LPS have achieved impressive immunogenicity. The lack of animal models for Shigella infections has severely impaired the analysis of protection. Serum IgG antibodies specific for LPS appear to be important in protection, but the intracellular replication of the bacterium suggests that other mechanisms such as CD8+ T cells are important chapter (Vaccines against Infections Caused by Salmonella, Shigella, and Pathogenic Escherichia coli).
The variety of E. coli pathotypes and virulence determinants, combined with the diversity of diseases they induce, suggest that a single immunoprotective mucosal mechanism is unlikely to exist. Clearly, protection against disease can be conferred by mucosal immune responses generated against some of the key exotoxins and probably by antibodies specific for fimbrial adhesins. Immunity in mouse models of attaching/effacing bacteria suggests that serum antibodies are protective and require signals from CD4+ T cells for full effector function. This conclusion is supported by studies in cattle infected with EHEC.
The species Salmonella enterica contains a number of serovars that include pathogens of both humans and animals; these bacteria are frequently host specific and may cause different diseases in different hosts. Ingestion of various Salmonella serovars, such as Typhimurium, results in localized infections of the small intestine leading to gastroenteritis in humans, whereas ingestion of serovar Typhi results in systemic infection and enteric fever. Serovar Typhi infects only humans, and in this section, we discuss the mucosal immune responses against serovar Typhi, focusing on the responses in humans and in the mouse typhoid fever model.
Studies with human volunteers have shown that the infectious dose of serovar Typhi ranges from 1,000 to 1 million organisms (67), but infectious doses of S. enterica in food-borne infections may be as low as three to six organisms (14, 60). The importance of nonimmune defense mechanisms against serovar Typhi infection has been investigated to a certain extent. It is well known that gastric hypochlorhydria increases the risk of infection with nontyphoidal salmonellosis and the severity of disease (64) and lowers the infective dose of serovar Typhi as well. The importance of the normal bowel flora for protection against colonization by serovar Typhi has been well established (67). Treatment of human volunteers with streptomycin, which kills Bacteroides and lactobacilli indirectly through killing facultative normal flora and alteration of redox conditions, significantly reduced the infectious dose of serovar Typhi. Similarly, treatment of mice with streptomycin renders the animals more susceptible to infection with serovar Typhimurium (5).
Histological analysis of small-intestine biopsies shows that a mononuclear cell infiltrate seems responsible for the enteritis observed early after infection with serovar Typhi (13, 166). Results of numerous in vitro studies have suggested that the early inflammatory response is due to secretion of inflammatory mediators by infected epithelial cells, such as tumor necrosis factor alpha (TNF-α) and IL-6 (40). Exposure of epithelial cells may also result in the production of chemokines, resulting in the migration of neutrophils, macrophages, dendritic cells, and T lymphocytes to the site of infection (40). Of interest, however, is a recent finding that the serovar Typhi Vi capsule may inhibit signaling in epithelial cells, at least in an in vitro model system (159), which may explain the lower levels of enteritis induced by serovar Typhi than by serovar Typhimurium.
Studies in mice have demonstrated that Salmonella may exploit the inflammatory response to establish an infection (118). Colonization of mucosal tissues and systemic dissemination was significantly reduced, or absent, in caspase-1 knockout mice, which cannot cleave proinflammatory cytokines IL-18 and IL-1β into their bioactive form. Seemingly paradoxically though, IFN-γ and IL-1 are known to play important protective roles during later stages of infection (40, 110).
Infection with serovar Typhi induces systemic and mucosal humoral and cell-mediated immune responses; these responses do not offer complete protection against relapse or reinfection (39, 107), suggesting that serovar Typhi is somehow able to manipulate the effector arm(s) of the immune system. Patients with typhoid fever have high titers of circulating IgM, IgG, and IgA LPS-specific antibodies, and anti-flagellar IgG antibodies, as well as antibodies against Vi antigen, porins, and outer membrane proteins (67, 68, 133). A two-year study among typhoid patients in Thailand described systemic and intestinal antibody responses to serovar Typhi (153). This study showed that serum LPS O-antigen and flagellar H-antigen-specific antibodies, as measured by Widal agglutination tests, peaked around 4 weeks after onset of disease and then rapidly declined; O-antigen-specific agglutinins persisted for 16 weeks, whereas H-antigen-specific agglutinins were detectable for 36 weeks. serovar Typhi protein antigen-specific serum antibodies were measured by enzyme-linked immunosorbent assay (ELISA). This analysis showed that IgM responses were detectable for up to 16 weeks after onset of disease, while IgG responses were not only higher than IgM responses but also lasted longer and were detectable up to 2 years after infection. The SIgA response to protein antigens was measured by ELISA using intestinal lavage samples. Although the peak of the response occurred early after infection (4 weeks), the SIgA was detectable for 2 years after onset of disease.
In endemic regions in Vietnam, however, anti-LPS and anti-flagellum titers are also elevated in noninfected persons (68). There seems to be no clear correlation between high titers of circulating serovar Typhi-specific antibodies and clearance of bacteria and/or protection against reinfection (39, 67, 189), although results from vaccine trial studies have suggested that the best correlate of protection is the antibody response to the LPS O9,12 antigen (93, 185). In addition, it has been shown that MHC-II and -III alleles HLA-DRB10301/6/8, HLA-DQB10201-3, and TNFA2(-308) are associated with susceptibility to typhoid fever in patients in Vietnam (38). On the other hand, resistance to typhoid was associated with HLA-DRB104, HLA-DQB10401/2, and TNFA1(-308). No clear association has been established between HIV infection and the incidence of typhoid fever (28, 105). Individuals infected with HIV, however, frequently suffer from (recurrent) nontyphoidal salmonellosis, as do individuals with limited capacity to produce, or respond to, IL-12 and/or IFN-γ (4, 30, 52, 55, 56, 141). Together, these studies suggest that the cell-mediated immune response to Salmonella spp. is important in the control of infection, but it may be more effective against nontyphoidal serovars.
Much of what is known about specific cellular immune responses to serovar Typhi has been learned from studies in which human volunteers were immunized with live attenuated oral vaccines, such as Ty21a. Ty21a is a galE, Vi-negative serovar Typhi strain that was created by chemical mutagenesis and is currently the only licensed live oral typhoid vaccine. Clinical trials have demonstrated that the efficacy of the vaccine varies from 67% to 96%, depending on the form in which the vaccine is taken (Table 3). Numerous studies have been performed in which human volunteers were vaccinated via a mucosal route (e.g.. orally or rectally) with the Ty21a vaccine, and immune parameters detected in these studies are generally considered important markers of protection against serovar Typhi infection.
Table 3Efficacy of a live oral vaccine (Ty21a) against typhoid fever |
After oral vaccination with Ty21a, O-antigen-specific serum IgG and IgA responses, as well as circulating gut-derived IgA antibody-secreting cells (ASC), IgG and IgM ASC can be detected among peripheral blood mononuclear cells (PBMCs), and the presence of these responses broadly correlates with protection against infection (185, 186). In addition, intestinal SIgA specific for LPS O-antigen can be detected in fecal samples (128) and jejunal fluid (47). Similar results have been obtained by using other live attenuated vaccine strains. After administration of a single oral dose of 5 × 109 CFU CVD 908-htrA (ΔaroC ΔaroD ΔhtrA serovar Typhi), serum antibody responses against LPS and flagellar H antigen, as well as ASC producing anti-LPS IgA, IgG, and IgM, were detectable (170, 171). Using a serovar Typhi Δcya Δ(crp-cdt) Δasd strain, Nardelli-Haefliger et al. showed that a single oral dose induced seroconversion against LPS in six of seven volunteers, and SIgA was detectable in mucosal secretions of four volunteers (126).
Some studies have shown that successful induction of intestinal SIgA responses following secondary immunization with Ty21a inversely correlates with the presence of O-specific serum antibodies, and a booster dose 14 months after a primary oral immunization fails to significantly increase ASC responses measured in PBMCs (186). Similar findings were observed by Forrest, who reported that the intestinal antibody response to Ty21a is reduced in individuals with elevated specific IgA levels before vaccination (45). In contrast, parenteral immunization (which does not induce IgA responses) before an oral booster immunization does not affect secondary ASC responses (46). Another study showed that a booster after 3.5 years instead of 2.5 years after the primary vaccination is more effective in increasing anti-LPS antibody levels in serum and stool samples (34, 87). These investigators concluded that the local mucosal immune response, which is present for at least 2.5 years after oral immunization with Ty21a, restricts colonization of the gastrointestinal tissues by the vaccine organism, resulting in a reduced booster effect. These studies also showed that antigen-specific T-lymphocyte proliferative responses are still detectable in these individuals (186). Other studies, however, have suggested that seroconversion following immunization with the serovar Typhi Δcya Δ(crp-cdt) Δasd strain is increased in volunteers with higher pre-immune anti-LPS IgG titers (126).
The explanation for the discrepancy between these studies is not clear, but may be the different methods that were used to measure pre-existing and post-immunization immunity, since no clear correlation has been established between the number of circulating ASC and antibody levels in serum or intestinal lavage samples, between serum IgA and fecal SIgA levels, or between fecal SIgA (50) and intestinal SIgA levels. In addition, the effect of pre-existing intestinal immunity may affect individuals living in endemic areas in a different way than persons living in typhoid-endemic areas. Finally, other immune effector mechanisms, such as T-lymphocyte responses, have not been intensively investigated in these studies and may well play a major role in exclusion of vaccine and/or pathogenic organisms. Note that the presence of serum anti-flagella antibodies in nonvaccinated controls is associated with a lower attack rate (67), and a vaccine without H antigen gave poor results in Egyptian children (189). From these observations it can be concluded that anti-flagellar antibodies, and especially those secreted into the mucosa, are important in protection against colonization and/or invasion of the epithelial barrier by salmonellae. These observations are also important when considering the natural history of disease: chronic carriers of serovar Typhi shed large numbers of bacteria from their gall bladder but do not show any symptoms of disease. It has been suggested that these individuals may have strong intestinal immune responses that protect the gut from invasion. The same could be true for people living in areas where the disease is endemic, who are continuously exposed to the bacterium but do not develop disease.
More recent studies have investigated mucosal humoral immune responses at a different level. In Ty21a-vaccinated volunteers, antibodies specific for homing receptor α4β7 were used to identify antigen-specific ASCs in PBMCs that are destined to migrate to the gut mucosa. These studies showed that following oral vaccination, ASC responses peak in PBMCs at about 1 week after immunization, and 99% of these ASCs express α4β7. In contrast, the homing receptor for peripheral lymph nodes, L-selectin, was expressed on only 22% of ASCs (80, 81). These results suggest that antigen-specific ASCs induced by oral vaccination with Ty21a migrate back to the gastrointestinal effector sites within 7 to 10 days after activation, where they secrete the antibodies to protect local tissues.
Several studies have reported on the T-lymphocyte responses to serovar Typhi antigens in response to oral vaccination with live attenuated vaccine strains. Proliferation of both CD4+ and CD8+ T lymphocytes can be detected following in vitro restimulation with either serovar Typhi-infected autologous lymphoblasts, whole inactivated serovar Typhi or flagellar antigens (99, 150, 151, 169, 171, 186). CD4+ T lymphocyte responses are of the "T helper 1" (Th1) type; restimulation of antigen-specific CD4+ T lymphocytes results in the production of IFN-γ in the absence of IL-4 secretion. The study by Lundin et al. showed that the CD4+ and CD8+ peripheral T lymphocytes of Ty21a-vaccinated individuals that responded to in vitro restimulation by production of IFN-γ were memory T cells (CD45RA-), most of which expressed the gut homing integrin β7 at high levels (99). The responses of these cells circulating in the bloodstream peaked around day 7 after vaccination. Other studies have further characterized the CD8+ T lymphocyte response to oral vaccination with an serovar Typhi vaccine (150). These investigations have shown that PBMCs from Ty21a-vaccinated volunteers can lyse serovar Typhi-infected autologous lymphoblasts after an 8-day in vitro culture with APCs, and this lysis is mediated by CD3+ CD8+ CD56− cells. These CD8+ cytolytic T lymphocytes also produce IFN-γ in response to culture with serovar Typhi-infected blasts, as demonstrated by ELISPOT and intracellular staining. The serovar Typhi-specific CD8+ T-lymphocyte responses can be detected up to 2 years after vaccination in peripheral blood. Similar findings were observed after vaccination with CVD908-htrA (151). If the immune responses elicited with attenuated serovar Typhi strains reflect the immune responses triggered by natural infection with serovar Typhi, these studies suggest that serovar Typhi infection induces the activation of both CD4+ and CD8+ T lymphocytes that circulate in the periphery and have the capacity to migrate into the gastrointestinal mucosa via the α4β7 homing receptor. An important function of these lymphocytes is the secretion of cytokines such as IFN-γ, which in turn may help activate macrophages to kill the bacteria. An additional role may be participation in antibody-dependent cellular cytotoxicity (172).
One of the most severe complications associated with typhoid fever is gastrointestinal perforation, which usually occurs in the terminal ileum during the later stages of infection and requires surgical as well as antimicrobial treatment (13). The pathogenesis of gastrointestinal perforation is not well understood and may partly result from the local immune response in GIT (23, 43). A recent publication reported on the histological analysis of biopsies taken from patients with suspected intestinal perforation associated with typhoid fever in the endemic Mekong Delta region of Vietnam (127). The major findings of this study were that a discrete, chronic inflammatory infiltrate was evident around the site of perforation with mild pathological changes of the mucosa, whereas severe inflammation was observed in deeper tissues, i.e., the submucosa and muscularis. In these tissues, CD68/PGM1+ macrophages and CD3+ T lymphocytes dominated the infiltrate, and the pathological changes were indicative of TNF-α-mediated damage. It was also noted that bacteria were rarely detected (either by histology or culture) in the biopsies, suggesting that a limited number of bacteria in the Peyer’s patches were able to evoke an exaggerated immune response in these patients. The authors suggested that this inappropriate response may have resulted from prior exposure to serovar Typhi, consistent with the observation that perforation usually occurs during second or third encounters with serovar Typhi (23).
Oral infection of mice with serovar Typhimurium results in a systemic disease, and this model is frequently used as an experimental animal model to study the innate and acquired immune responses during typhoid fever. Extrapolation from the immunology of murine typhoid has been complicated, however, by the observation that inbred strains of mice vary in their sensitivity to serovar Typhimurium infection, from relatively resistant (oral LD50 ≥ 108 bacteria or ItyR) to highly sensitive (oral LD50 ≤ 104 bacteria or ItyS) (142). This natural resistance is mediated by a single locus on chromosome 1 called Nramp1 (also referred to as Slc11a1, previously called Ity), which also confers sensitivity or resistance to other intracellular pathogens (Mycobacterium spp., Leishmania spp., and Salmonella spp.) (184). The natural resistance-associated macrophage protein (Nramp1) is almost exclusively expressed by macrophages, and susceptibility or resistance to murine serovar Typhimurium infection is determined by a mutation in Nramp1 that results in a single amino acid substitution (183) (see elsewhere in EcoSal). Studies in humans, however, have failed to determine a link between polymorphic NRAMP1 alleles and susceptibility to typhoid fever (36). To overcome the lethality of serovar Typhimurium infection in susceptible mice, many researchers have used attenuated strains of serovar Typhimurium to experimentally infect mice.
Serovar Typhimurium uses a Salmonella pathogenicity island-1 (SPI-1)-encoded mechanism to preferentially invade the murine gastrointestinal barrier through M cells (51, 78, 79), although the bacteria may also invade epithelial cells (173). Recent studies have shown that dendritic cells can phagocytose Salmonella from the intestinal lumen and transport it across the epithelial barrier (147), and that migrating CD18+ phagocytic cells, possibly dendritic cells, are involved in systemic spread of the bacteria (182). Other studies have also revealed that attenuated serovar Typhimurium strains can persist within dendritic cells in the PPs (65, 66, 108), and since it has been shown that Salmonella-infected dendritic cells can stimulate antigen-specific T lymphocytes (reviewed in reference 196), it is very likely that the dendritic cells in the PPs are involved in the induction of anti-Salmonella immunity. Note that the studies by Vazquez-Torres et al. showed that systemic infection as a consequence of phagocytosis of Salmonella by CD18+ cells is SPI1 independent and results in the induction of systemic IgG responses but not mucosal IgA responses (182). This finding implies that induction of Salmonella-specific mucosal immune responses requires local (i.e., in the PPs) interaction of APCs with antigen-specific lymphocytes. In vitro experiments have shown that epithelial cells are stimulated by Salmonella flagellin to produce the chemokine CCL20, which triggered the migration of immature dendritic cells (160). This may be one mechanism by which dendritic cells are attracted to a site of Salmonella invasion. In turn, Salmonella-infected dendritic cells may also produce inflammatory and chemoattractive cytokines to attract more immature dendritic cells, T lymphocytes, and other immune effector cells to help combat the infection (50, 108).
In recent reports, the interaction between Salmonella and dendritic cells was examined more closely. By using confocal microscopy, it was demonstrated that, following oral infection of mice, not only dendritic cells in the PPs but also those in the mesenteric lymph nodes are the main cell population interacting with serovar Typhimurium (25). Furthermore, it was demonstrated that serovar Typhimurium is able to interfere with MHC-I- and -II-mediated antigen presentation via a SPI-2-mediated mechanism (25, 176). It will be of great interest to determine which specific Salmonella genes are involved in downregulation of antigen presentation by the infected dendritic cells.
Several studies have used mice that lack one or more subsets of T lymphocytes and have demonstrated that immunity provided by Salmonella-specific T lymphocytes is indispensable for the control of serovar Typhimurium infections in mice (62, 111, 163, 190). These studies have pointed to a major role for IFN-γ-producing CD4+ T lymphocytes that express the αβTcR in control of the infection and protection against reinfection.
In this section, we focus particularly on the activation of Salmonella-specific T cells in the gastrointestinal tissues of mice, which have been studied in a number of ways. By using adoptive transfer of fluorescently labeled antigen-specific CD4+ T lymphocytes, the activation of these T lymphocytes following infection with serovar Typhimurium has been followed in some very elegant studies. Using a recombinant, attenuated serovar Typhimurium (ΔaroA) strain, Bumann showed that adoptively transferred T lymphocytes specific for the recombinant antigen accumulate in the PPs from 3 days after infection, peaking at about 8 days after infection, and that these T lymphocytes express the early activation marker CD69 and are proliferating (19). Histological analysis of PPs demonstrated that the T lymphocytes localize in the interfollicular T-cell areas, whereas the Salmonella reside in the subepithelial dome and are not in direct contact with the T lymphocytes. Studies by McSorley et al. used T lymphocytes from transgenic mice that express a TcR specific for an immunodominant epitope of flagellin (113). After adoptive transfer, mice were infected with a virulent strain of serovar Typhimurium, and activation of flagellin-specific T lymphocytes was studied in the PPs, MLNs, spleen, and peripheral lymph nodes by using flow cytometry. These studies showed that flagellin-specific T lymphocytes were activated exclusively in the PPs and MLNs, and not in the spleen or peripheral lymph nodes, despite the presence of bacteria in the spleen. The activated T lymphocytes produced IL-2 and TNF-α, and some cells were producing IFN-γ or IL-4. Histological analysis showed that the adoptively transferred flagellin-specific T lymphocytes localized mainly in the T-lymphocyte-rich follicle of the PPs, MLNs, and spleen.
Following infection with serovar Typhimurium, early activation of the T lymphocytes occurs in the T-cell-rich areas of the PPs and MLNs, and the activated T lymphocytes then migrate to the B-lymphocyte follicles, where they help B lymphocytes mature into antigen-specific antibody-producing cells. This study failed to demonstrate migration of activated flagellin-specific T lymphocytes to the lamina propria. However, presumably because the mice became ill from the lethal infection, this experiment had a short time span (5 days); it is possible that T lymphocytes would have migrated to the lamina propria at later time points. An alternative explanation is that bacteria present in the lamina propria, e.g., those that invaded the epithelial cells, may no longer be flagellated so that flagellin-specific T lymphocytes would not migrate into the lamina propria.
After oral infection of mice with serovar Typhimurium, specific T-lymphocyte responses can be detected in the PPs and MLNs. Salmonella-specific CD4+ T lymphocytes produce IFN-γ and IL-4 in response to in vitro restimulation, and responses peak about 3 to 4 weeks after infection (54). CD4+ T lymphocytes isolated from the lamina propria produce relatively more IFN-γ than IL-4, and these responses are detectable for at least 9 weeks after infection. Another study used mRNA analysis to determine cytokine environment in PPs and MLNs after immunization with attenuated serovar Typhimurium strains and demonstrated the expression of IL-4, IL- 6, IL-7, IL-12, and IFN-γ mRNA at increasing levels from day 1 to day 7 after immunization (83).
In many studies recombinant attenuated serovar Typhimurium strains were used to immunize mice, and T-lymphocyte responses specific for the recombinant antigen were examined. When measured 4 to 6 weeks after infection, T-lymphocyte responses detected in the PPs and MLNs are dominated by production of IL-2 and IFN-γ, but IL-6 and IL-10 secretion can also be detected (181). Another study demonstrated that T-lymphocyte responses in the PPs specific for recombinant antigen change during the course of the infection. Early after infection (i.e., 1 week), T-lymphocyte responses are mixed, and production of IL-4 and IL-5 as well as IFN-γ and IL-2 by the T lymphocytes can be detected, whereas IL-4 and IL-5 are, as reported in other studies, undetectable later (135).
Some studies have indicated that serovar Typhimurium strains that harbor different defined mutations in virulence genes may elicit altered T-lymphocyte responses, possibly as a result of changes in the inflammatory response (37, 114, 180). In addition, the T-lymphocyte response detected against recombinant antigens may not necessarily be the same as the immune response against the bacterium itself (135, 181). It would therefore be more informative to evaluate mucosal T-lymphocyte responses in resistant mice (i.e., Nramp+/+) infected with a virulent serovar Typhimurium strain to more accurately reflect responses during "natural" serovar Typhimurium infections. Mittrucker et al. recently studied T-lymphocyte responses in (C57BL/6 × Sv129) F1 mice (Nramp+/−), but these experiments were limited to T-lymphocyte responses in the spleen following intravenous infection with virulent strain SL1344 (117).
Immunization with serovar Typhimurium also results in the activation of antigen-specific CD8+ cytotoxic T-lymphocyte responses, which can be detected in mucosal tissues and in the spleen (95, 177, 193). One study has reported that some CD8+ T lymphocytes induced by immunization with serovar Typhimurium may recognize antigen presented by the nonclassical MHC-Ib molecule Qa-1b, and showed that the CD8+ T lymphocytes are also able to recognize cells infected with other Salmonella strains (97), suggesting that they may be specific for highly conserved bacterial proteins. CD8+ T lymphocytes may contribute to control of Salmonella infection by lysis of infected host cells, which would render the intracellular bacterium susceptible to killing by macrophages or opsonization with antibody. In addition, CD8+ T lymphocytes may be an important source of IFN-γ.
Circulating T lymphocytes activated in PPs or MLNs are recruited to the lamina propria of the gut via interaction of mucosal homing receptor α4β7 and MadCAM-1, which is expressed on endothelial cells in the PPs, but MadCAM-1 can also bind L-selectin (CD62L), which in general is considered to be a peripheral homing receptor (123). Mucosal immune responses against Salmonella depend on the expression of L-selectin, as has been demonstrated in L-selectin knockout mice (136). Following immunization of these mice with attenuated serovar Typhimurium, SIgA responses were undetectable in the feces, but serum IgG responses were elevated compared with L-selectin+/+ mice. Despite the presence of normal numbers of CD4+ T lymphocytes, the T lymphocytes isolated from PPs of L-selectin−/− mice did not produce cytokines (IFN-γ, IL-4, IL-6, IL-10) upon restimulation in vitro, and splenic T-lymphocyte responses were reduced. Unfortunately, the investigators did not study the Salmonella-specific responses of lamina propria lymphocytes. The L-selectin−/− mice were completely protected against challenge infection with a lethal dose of a virulent serovar Typhimurium strain, suggesting that although the induction of mucosal immunity relies on expression of L-selectin, the mice are still protected against challenge infection in the absence of these responses.
Taken together, these results provide evidence that, shortly after oral infection of mice, Salmonella-specific T lymphocytes are activated in the PPs and MLNs and migrate to the B-lymphocyte-rich areas in the PPs and MLNs. Activation of Salmonella-specific T cells results in the production of T helper 1 and T helper 2 type cytokines, which in turn results in the activation of Salmonella-specific B lymphocytes and ultimately the production of Salmonella-specific antibodies, and which may help activate microbicidal mechanisms of macrophages.
Infection of mice via the oral route with serovar Typhimurium results in the induction of intestinal Salmonella-specific SIgA and systemic antibody responses (3, 179, 181). These responses can be measured in the serum as well as intestinal washes and fecal samples, and antibody levels in feces remain high for up to 9 weeks after a single oral immunization (179). LPS-specific ACS can be detected in the PPs, MLNs, spleen, and lamina propria, and responses peak at about 25 to 30 days after immunization with attenuated serovar Typhimurium (3).
It is commonly believed that the Salmonella-specific SIgA antibodies protect the GIT from bacterial invasion. Indeed, in vitro experiments have shown that a monoclonal polymeric anti-Salmonella LPS IgA is able to inhibit adherence and invasion of epithelial cells by serovar Typhimurium (116). Further studies have also demonstrated that mice carrying a "back-pack hybridoma tumor" secreting a monoclonal Salmonella LPS-specific IgA, are protected from infection with Salmonella via the oral route (115). However, our own experiments have shown that protection against invasion of the GIT by virulent serovar Typhimurium can occur independent of Salmonella-specific secretory antibody responses (179). Mice were orally immunized with an attenuated serovar Typhimurium strain known to elicit protective immunity (ΔaroAD), and 8 weeks after vaccination, when Salmonella-specific SIgA responses were still high, the early invasion (i.e., 6 h after oral infection) of PPs by virulent serovar Typhimurium was compared between naïve and immune animals. In these experiments, we were unable to detect a difference in the number of bacteria invading PP of naïve mice compared with immune animals, suggesting that the antigen-specific SIgA did not protect the epithelium from invasion by serovar Typhimurium (107 CFU). In addition, bacterial invasion was compared between normal and pIgR knockout (pIgR−/−) mice, which are unable to bind polymeric antibodies at the basolateral side of epithelial cells because of a mutation in the pIgR and are therefore deficient in secretory antibodies (76, 178). Serovar Typhimurium invasion of the gut was similar in vaccinated normal and pIgR−/− mice. Furthermore, vaccinated normal and pIgR−/− mice were equally protected against a challenge infection with 107 CFU, suggesting that the secretory immune response is not an absolute requirement for protection of the gastrointestinal epithelium against salmonellae (179). Given that serovar Typhimurium breaches the epithelium, other immune effectors may provide sufficient protection to prevent disease.
Using a "natural" infection model, we recently demonstrated that polyreactive secretory antibodies (i.e., those present in naïve animals) are of major importance in protection against serovar Typhimurium infection (192). To mimic the natural fecal-oral route of infection, mice infected with a virulent strain of serovar Typhimurium were cohoused with naïve animals. The infection was monitored in all mice by enumerating the number of serovar Typhimurium shed in the feces. These experiments showed that under such circumstances, naïve pIgR−/− mice were profoundly sensitive to infection, resulting in 50% death of animals, whereas only one of six normal mice acquired the infection, and none died. Moreover, the infected pIgR−/− mice were shedding significantly higher numbers of bacteria, which more readily infected other animals. These results suggest that the mucosal secretory immune system provides an innate barrier that protects the mammalian host against infection and, perhaps more importantly, limits the impact of disease on the herd. Indeed, one study has reported that total intestinal IgA levels were decreased in patients with typhoid fever compared with control individuals, and the authors of this study suggested that the lower IgA levels rendered the patients more sensitive to infection by serovar Typhi (84).
Not all Salmonella infections result in systemic dissemination of the bacteria and enteric fever; infection of humans with nontyphoidal salmonellae may be restricted to the gastrointestinal tissues, and bacterial replication may not occur beyond the MLNs, in which case the pathology is usually limited to enteritis.
Invasion of the epithelial cell barrier by serovar Typhimurium results in an inflammatory response in the underlying tissue. Early infiltration and transmigration of epithelium by polymorphonuclear cells (PMNs) may create abscesses in the crypts and fluid retention that can result in diarrhea. The mouse model has been of limited use to study Salmonella-induced gastroenteritis, since infection with serovar Typhimurium does not induce diarrhea in mice and only a limited, mild inflammatory response. For this reason, many researchers have used either in vitro models with polarized epithelial cells or other animal models to study responses to Salmonella-induced enteritis (reviewed in references 71, 132, 152, and 188).
A vast number of in vitro studies, using various human gut epithelium-derived cell lines, have been reported in the literature, and these experiments have provided insight into the mechanisms used by Salmonella to evoke an inflammatory response from epithelial cells, and have elucidated many of the inflammatory mediators involved in Salmonella-induced enteritis (for recent reviews refer to references 40, 71, and 152) (see elsewhere in EcoSal). These studies have revealed that invasion of epithelial cells by salmonellae depends on SPI-1-encoded genes (51), and in addition, that secretory and inflammatory responses are activated by effector proteins delivered by SPI-1-encoded TTSS (53). Exposure of epithelial cells to salmonellae induces the release of a number of chemokines that attract PMNs, such as IL-8, GROα, GROβ, GROγ, and ENA-78 (40). Other chemokines produced may attract monocytes (RANTES, MCP-1, MIP-1β, and MIP-3α), immature dendritic cells (MIP-3α) or subsets of T lymphocytes (IP-10, Mig, I-TAC, MIP-3α) (reviewed in reference 40). Other in vitro studies have revealed that while most of the aforementioned chemoattractants are released on the basolateral side of the epithelial cells, the pathogen-elicited epithelial chemokine is secreted from the apical side of the cells, possibly attracting PMNs to the intestinal lumen (112). By releasing such a mixture of chemokines and cytokines, the epithelial cells may attract many immune effector cells to the side of infection, and even regulate their responses.
As in humans, infection of a large number of animals with serovar Typhimurium induces localized enteric infections in the gut tissue accompanied by diarrhea. Injection of salmonellae into ligated ileal loops from rabbits or calves induces intestinal inflammation and fluid retention, and this experimental model has frequently been used to study the pathogenesis of Salmonella-induced gastroenteritis and to confirm observations from tissue culture experiments. Although the pathology observed in ligated loops mimics that seen following infection via the natural oral route, the model can only be used to study early events following infection. Results obtained with this model have been reviewed in detail elsewhere (152, 188); a number of studies have validated the secretion of immunomodulatory chemokines and cytokines by intestinal epithelial cells following injection of salmonellae into ileal loops.
Unlike the acute inflammatory responses, the specific adaptive immune responses to nontyphoidal salmonellae have not been widely studied. It is well known, however, that in immunocompromised individuals, nontyphoidal salmonellosis may cause bacteremia and sometimes more serious disease, such as endovascular infections (64). Recent reports have pointed to a high incidence of nontyphoidal salmonellosis among HIV-infected individuals. A study from Spain demonstrated that nearly 60% of patients presenting with a relapse of Salmonella septicemia after treatment were infected with HIV (52). In another study among patients in Malawi, Africa, it was shown that 77 of 78 adult cases of nontyphoidal Salmonella bacteremia were HIV positive, which was associated with a high mortality (47%, increasing to 77% at 1 year) and recurrence rate (43%) (55). The results from this study also demonstrated that recurrent infections were the result of recrudescence rather than reinfection. Similar findings were obtained in a study among Malawian children who presented with nontyphoidal Salmonella bacteremia (56). Other clinical studies have demonstrated that patients with a deficiency in the IL-12p40 subunit and patients harboring a genetic mutation in the IL-12 receptor β1 chain (IL12Rβ1) are deficient in IL12R signaling and production of IFN-γ and suffer from severe recurrent infections with Salmonella spp. (4, 30, 141). These findings indicate that adaptive cellular immune responses are of major importance for control and eradication of nontyphoidal Salmonella infections.
In their study, Hindle et al. compared immune responses induced with an attenuated serovar Typhimurium strain (WT05) and an attenuated serovar Typhi strain (ZH9), each harboring identical mutations in the aroC and ssaV genes (63). In individuals who received the highest dose of the vaccine, an acute-phase response and elevated IL-6 levels were measurable in the serum, indicative of inflammatory responses. Salmonella LPS-specific IgA and IgG ASCs were detectable in PBMCs, with the highest numbers detected 7 days after immunization. Anti-O-antigen IgA and IgG responses were also measurable in the serum by ELISA. No correlation was obvious between the length of shedding (in days) of the vaccine organism and the measured levels of mucosal antibodies (IgA). Together, these results suggest that infection with serovar Typhimurium induces an acute inflammatory response followed by antigen-specific immune responses that are essential for control of the bacterium.
Results of studies with human volunteers have suggested that mucosal humoral and cellular immune responses are induced following oral infection with serovar Typhi or vaccination with an attenuated live serovar Typhi vaccine, and that these responses may be involved at least in part in protection against (re)infection. Vaccine studies have shown that a second immunization at a time when mucosal immune responses are still high does not result in a boost of antigen-specific antibody responses, suggesting that the local immune responses suppress the colonization of tissues by the vaccine organism. On the other hand, mucosal immunity may also be to blame for pathology observed in the gut (i.e., intestinal perforation) during serovar Typhi infections. Susceptibility to serovar Typhi does not seem to be increased in individuals with compromised cellular immunity (e.g., HIV/AIDS, IL12-R, or IFNγ-R deficiency). Instead, these individuals suffer from recurrent septicemic serovar Typhimurium infections, suggesting that local cellular immune responses are of major importance in preventing the spread of nontyphoidal salmonellae from MLNs.
Studies in mice have shown that mucosal antibodies can protect against invasion of the gut epithelium by serovar Typhimurium. However, studies in specific gene knockout mice that lack secretory antibodies have suggested that the presence of Salmonella-specific secretory antibodies in the gut lumen is not essential for protection against reinfection. In conclusion, local cellular immune responses may be more important than humoral responses for protection against infection with salmonellae.
We acknowledge the financial support of the Australian Government-funded Cooperative Research Centre Program and the National Health and Medical Research Council of Australia. O.L.C.W. and R.A.S. are members of the Australian Bacterial Pathogenesis Program. This review is dedicated to the late Dr. Bruce Stocker, who was an inspiration to a generation of molecular microbiologists interested in pragmatic applications of bacterial molecular biology.
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