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Salmonella flagellin, a microbial target of the innate and adaptive immune system
Immunology Letters 101 (2005) 117–122
Review
Salmonella flagellin, a microbial target of the innate and adaptive immune system Rosa Maria Salazar-Gonzalez, Stephen J. McSorley ∗ Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030-1319, USA Received 9 May 2005; accepted 13 May 2005 Available online 6 June 2005
Abstract Bacterial flagellins are important components of the motility apparatus used by many microbial pathogens. These proteins are also targets of the innate and adaptive immune response of the host during infection and autoimmune disease. Flagellin interacts with TLR-5 and leads to the generation of a pro-inflammatory response and activation of host dendritic cells in vivo. Furthermore, flagellin is recognized by antibody and CD4 T cells responses during Salmonella infection. Here, we review recent developments in the understanding of flagellin interactions with the host immune system. © 2005 Elsevier B.V. All rights reserved. Keywords: Flagellin; Salmonella; Innate immunity
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and function of flagellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of flagella in Salmonella pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural interactions between flagellin and TLR-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pro-inflammatory activity of flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flagellin and inflammatory disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adjuvant activity of flagellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adaptive immune response to flagellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bacterial flagellins have been studied for decades, due to the importance of these proteins in bacterial motility, and the complex nature of flagellar gene expression. However, renewed interest in flagellins arises from the recent understanding that these molecules are specifically recognized by ∗
Corresponding author. Tel.: +1 860 679 8785; fax: +1 860 679 1868. E-mail address: [email protected] (S.J. McSorley).
0165-2478/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2005.05.004
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the innate immune system. Therefore, these microbial products are members of a growing family of pathogen-associated molecule patterns (PAMPs), molecules that are used by the mammalian host to detect invasion by microbial pathogens. Recent work in our laboratory, and others, points to the fact that Salmonella flagellin is a target of both the innate and adaptive immune response during murine typhoid. Here, we discuss the interaction of bacterial flagellins with the innate and adaptive immune system, placing particular emphasis on the role of this interaction during Salmonella infection.
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2. Structure and function of flagellin Flagellin is the major protein constituent of bacterial flagella, complex surface appendages that are involved in bacterial locomotion. More than 50 genes are known to be involved in the regulated expression and function of the flagellum, implying that motility and chemotaxis are critically important for bacterial survival [1]. Flagella are part of the sensory machinery that allow bacteria to either respond to chemotactic stimuli, or simply avoid an unfavorable environment, such as extreme pH or saline concentrations. The structure is a self-assembling sub-system, consisting of a long helical filament emerging from a flexible hook, connected to a basal body that is anchored in the inner and outer cell membrane [1]. When in use, the flagellum exhibits a random pattern of movement characterized by two different turns, a counterclockwise rotation, when the bacteria packs all filaments into one structure, and an alternative clockwise rotation that causes separation of the filaments and chaotic movement of the bacteria [2]. Polymers of a single protein, flagellin, compose the 11 filaments of each flagellum. Salmonella flagellin is comprised of 494 amino acids and distinct domains have been described, based on homology between different Salmonella serovars [3–5]. Both the amino- and carboxy-terminus are well conserved among Salmonella serovars, while the central portion displays more diversity, one particular region being termed the “hypervariable region”. Recent analysis of the crystal structure of a Salmonella flagellin fragment revealed that the protein filament is folded back upon itself such that the amino- and carboxy-termini are physically located in close proximity to each other, within the central portion of the filament, and are likely involved in determining the repeating 3-D structure [6,7]. In contrast, the central portion of flagellin is exposed on the outside of the filament, providing a simple explanation for the fact that antibody responses tend to be targeted to this region [8].
3. The role of flagella in Salmonella pathogenesis Mucosal infection with bacterial strains that are genetically deficient in flagellin or flagellar-associated genes have demonstrated an obligatory role for the flagella in bacterial adhesion to epithelial surfaces, colonization, biofilm formation, and invasion of host tissues. For example, a flagellindeficient strain of Pseudomonas aeruginosa was found to be severely attenuated compared to the flagellated parental strain [9]. Histological analysis in this model demonstrated that flagellin-deficient strains cause a localized infection foci, in contrast to the typical spreading of infection observed during wild-type Pseudomonas pulmonary infection [9]. In another model system, expression of flagella is required for the persistence of Helicobacter pylori following infection of piglets [10]. For infection with Vibrio species, production of flagella is known to contribute to initial attachment of the bacteria
to the cell surface, biofilm formation and ultimate lethality [11,12]. Salmonella infection of inbred mouse strains is the best available laboratory model to study human typhoid fever [13] and flagellin expression is not normally considered to be a virulence factor [14,15]. However, recent experiments using rabbits uncovered a potential role for flagella in the initial interaction of Salmonella with M cells of the appendix [16]. Furthermore, flagellin expression is required for Salmonella invasiveness in a cell culture model, and for the induction of polymorphonuclear leukocyte infiltration using the calf intestine model of infection [15]. Therefore, although flagellin appears to be dispensable for Salmonella virulence in the mouse model, there are multiple lines of evidence to suggest that it is critically important for establishment of Salmonella infection in other species. The importance of flagellin expression to human typhoid fever remains to be established, but the available data from the models above suggest that it could play an important role in Salmonella typhi pathogenesis.
4. Structural interactions between flagellin and TLR-5 TLR-5 is expressed by epithelial cells, monocytes, and immature dendritic cells [17,18]. Initial identification of flagellin as the ligand for TLR-5 came from a study that isolated stimulatory components from Listeria culture supernatant proteins by HPLC [19]. Parallel studies examining the activation of epithelial cells in response to Salmonella also identified flagellin as a stimulatory ligand for TLR-5 [20]. Thus, TLR-5 can recognize flagellin that is produced by both Gram-positive and Gram-negative bacteria, providing a simple strategy for the host to respond to flagellated bacteria. In contrast to many other TLR ligands, the proteinaceous nature of flagellin has enabled a detailed study of the structural basis of flagellin–TLR-5 interactions. Two different groups have documented bioactivity of flagellin even after production in a eukaryotic expression system, demonstrating that the stimulatory capacity of flagellin is independent of other bacterial proteins or prokaryotic post-translational modifications [21,22]. Using defined deletion mutants of flagellin, two different groups initially identified two distinct sites required for bioactivity, the hypervariable region localized in domain 3, and the conserved amino- and carboxy-termini of the protein [23,24]. This discrepancy was resolved by more detailed mapping, which defined TLR-5 stimulatory activity within N-amino terminal residues 79–117 and 408–439 [22]. Thus, TLR-5 most likely recognizes a spatial area of flagellin comprising areas of the amino- and carboxy-terminus. The observation that the TLR-5 binding site of flagellin is most likely located within the central base of the filament is interesting since the residues needed for TLR-5 recognition are the same residues essential for the native structure of the flagellum and bacterial motility. Thus, the bacteria are unable to avoid host recognition by flagellin sequence muta-
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Fig. 1. Unresolved issues with regard to flagellin interactions with the immune system. Flagellin induces pro-inflammatory cytokine production and dendritic cell (DC) activation, eventually leading to CD4 T cell activation. However, several key aspects of this activity are unclear and are highlighted in the figure numerically. (1) What is the bioactive form of flagellin (polymer or oligomers) that activates pro-inflammatory activity? (2) Does flagellin induce DC maturation directly, via TLR-5 expressed on the DC, or (3) indirectly through cytokines produced by other cell types? (4) What type of adaptive immune response is induced by the adjuvant effect of flagellin, TH1, or TH2?
tion without compromising the beneficial aspects of bacterial motility. However, it is not yet clear how the internal structure of the flagellum filament would actually come in contact with TLR-5 in order to mediate pro-inflammatory activity in vivo (Fig. 1)? One possibility is that monomers or oligomers of flagellin are released from the filament in the hostile environment of the phagosome, or may be exposed during bacterial replication [25,26].
5. Pro-inflammatory activity of flagellin Initial studies using human blood mononuclear cells demonstrated that flagellin from different bacterial species stimulated the production of cytokines, such as TNF-␣ and IL-1- [25,27]. Furthermore, flagellin produced by S. typhi was found to induce synthesis of IL-6 and the antiinflammatory cytokine IL-10 [25,27–29]. More recent studies by Gewirtz et al. identified flagellin as the bacterial factor inducing IL-8 production following the interaction of Salmonella with human intestinal epithelial cells [30]. Interestingly, the activation of this inflammatory response was dependent upon the translocation of flagellin to the basolateral surface of the epithelial cells, where TLR5 is expressed [20,30]. Presumably, this requirement limits epithelial cell activation to pathogens that can translocate flagellin, and therefore, avoids TLR-5 ligation by the normal intestinal flora. Other studies have examined flagellin activation of human intestinal epithelial cell lines and noted the production of the chemokine CCL-20, and the subsequent migration of immature human DC in response to this stimu-
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lus [31]. In contrast to other studies, this report suggested that apical expression of TLR-5 was responsible for chemokine secretion [31]. In agreement with this notion, constitutive expression of TLR-5 has been reported on the apical surface of human primary IECs [17], and another recent study demonstrated that flagellin from commensal bacteria can induce TLR-5 activation on the apical surface of freshly isolated murine IECs [32]. Future studies are obviously required to resolve the issue of TLR-5 expression on the surface of epithelial cells and flagellin translocation (Fig. 1). If TLR-5 is expressed on the apical surface of epithelial cells, it remains unclear why a permanent inflammatory state is not induced in the intestinal mucosa in response to flagellated commensal bacterial. One possibility is that commensal microbes may also induce anti-inflammatory mediators or block inflammatory signaling pathways of epithelial cells [33,34]. TLR-5 is also highly expressed in the lung [35] and seems to play an important role in the defense against pathogens of the respiratory tract [36,37]. Interestingly, there is a correlation between a common human TLR-5 polymorphism and susceptibility to Legionnaires disease [38]. A singlepoint mutation to a stop codon at position 392 resulted in impaired responsiveness to flagellin in these patients [38]. Taken together, the available evidence suggests that expression of TLR-5 in the intestinal and respiratory mucosa is an important innate immune sensor for flagellated pathogens.
6. Flagellin and inflammatory disease Flagellin can induce expression of numerous proinflammatory mediators, such as TNF-␣, IL-1, IL-6, MIP-3␣, and iNOS, and therefore, may be involved in the development of bacterial-associated pathology. In support of this hypothesis, direct intratracheal instillation of 1 g of flagellin to mice resulted in an acute inflammatory process in the lung, involving neutrophil and macrophage infiltration and the detection of inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid [39]. Surprisingly, the administration of flagellin was more potent than LPS in the development of a lung inflammatory response [39], which could be due to low levels of TLR-4 in the lung, as described for intestinal epithelium [17]. It is possible, therefore, that chronic lung pathology caused by flagellated pathogens like Pseudomonas or Legionella is due in part to host responses to flagellin [37,38]. Analogous to endotoxin, flagellin is an effective mediator of systemic inflammation. Intravenous injection of mice with flagellin rapidly produced the typical pattern of cytokine, chemokine, and NO production, as well causing the clinic signs of septic shock; hypotension, respiratory distress, cyanosis, organ injury, and death [26,40]. Although it is not clear whether LPS or flagellin is more effective at causing toxic shock in mice [26,40,41], these data demonstrate that flagellin could be an important contributor to the inflammatory processes during bacterial sepsis.
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Crohn’s disease and ulcerative colitis are intestinal inflammatory disorders mediated by an abnormal immune response to commensal microbes. The identification of potential target antigens of the immune response has been a particular focus of research in this field. Recently, two different groups identified bacterial flagellin as an immunodominant antigen in Crohn’s disease [42,43]. High titers of anti-flagellin antibodies were detected in Crohn’s patients and colitic mice. Although these studies identified flagellin as a target of the adaptive immune response, it is also likely that the innate pro-inflammatory activity of bacterial flagellins can contribute to the pathology associated with this intestinal disease.
7. Adjuvant activity of flagellin As with other PAMPs, the function of flagellin as an adjuvant has been examined in detail. An initial report by our laboratory demonstrated that Salmonella flagellin was capable of enhancing antigen specific CD4+ T cell expansion and memory development in vivo [21]. This adjuvant effect of flagellin was dependent upon the ability to activate CD80/86 molecules expression and was completely blocked by administration of CTLA-4-Ig. Another interesting report noted that bacterial flagellin can directly activate human but not murine dendritic cells in vitro, suggesting that any adjuvant effect of flagellin in mice may be indirect and involve TLR-5 ligation on other cell types [44]. However, this latter observation has been challenged by another report that described direct flagellin activation of murine dendritic cells in vitro [45]. Differences in dendritic cell maturation or flagellin purification may explain these discrepancies between these studies. Preliminary data in our laboratory suggest that flagellin does not directly activate murine dendritic cells (Salazar and McSorley, unpublished data), and future experiments will hopefully resolve this important issue (Fig. 1). In our hands, and others, flagellin induced the development of antigen-specific Th1 response involving the production of IFN-␥ and not IL-4 upon recall stimulation [21,46,47]. These observations are consistent with a role for flagellin in contributing to the normal development of Th1 during murine Salmonella infection [48,49]. However, another report using flagellin as an adjuvant has described the generation of a Th2 CD4+ T cell response, finding IL-4, IL-13, and a typical Th2 antibody response after immunization [45]. This ability of flagellin to generate a Th2 response in vivo was corroborated by other group, who demonstrated convincingly that administration of soluble flagellin caused a Th2-like antibody response [50]. It remains unclear why flagellin can evoke such different T cell responses in different adjuvant model systems (Fig. 1). It seems likely that the purity of flagellin preparation, extent of polymerization, dose administered, and/or route of administration will explain these differences.
8. Adaptive immune response to flagellin As already noted above, bacterial flagellins are targets of the adaptive immune response during Crohn’s disease, and recent reports also suggest that flagellins may be important T and B cell targets during bacterial infection. Indeed, bacterial flagellins have been used for decades as target antigens to study the immune response in vivo. Elegant studies examining tolerance induction in rats and B cell antigen receptor recognition, made use of bacterial flagellin as a target antigen [51–53]. Furthermore, the polymeric structure and abundance of protein expressed by bacteria led to the use of flagellin as a carrier protein for B and T cell epitopes [54,55]. More recently, flagellin has been identified as a major target antigen of CD4+ T cell cells during murine and human Salmonella infection [56–58]. The poor definition of other targets of the adaptive immune response to Salmonella make evaluation of the importance of the flagellin-specific response difficult [59]. However, we do know that T cell responses to Salmonella flagellin occur rapidly after oral infection, and may contribute to vaccine-induced immunity [58,60]. Therefore, it seems likely that adaptive immune responses to flagellin are important in defense against bacterial infection.
9. Conclusion Bacterial flagellins are interesting microbial proteins that are targeted by both the innate and adaptive immune response. It seems likely that these proteins are critical to the development of protective immunity to bacterial infection, and the development of inflammatory conditions due to bacterial infection. The use of flagellins as part of vaccine or adjuvant preparations may lead to the development of novel live or sub-unit prophylactic treatments for several infectious diseases.
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Review
Salmonella flagellin, a microbial target of the innate and adaptive immune system Rosa Maria Salazar-Gonzalez, Stephen J. McSorley ∗ Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030-1319, USA Received 9 May 2005; accepted 13 May 2005 Available online 6 June 2005
Abstract Bacterial flagellins are important components of the motility apparatus used by many microbial pathogens. These proteins are also targets of the innate and adaptive immune response of the host during infection and autoimmune disease. Flagellin interacts with TLR-5 and leads to the generation of a pro-inflammatory response and activation of host dendritic cells in vivo. Furthermore, flagellin is recognized by antibody and CD4 T cells responses during Salmonella infection. Here, we review recent developments in the understanding of flagellin interactions with the host immune system. © 2005 Elsevier B.V. All rights reserved. Keywords: Flagellin; Salmonella; Innate immunity
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and function of flagellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of flagella in Salmonella pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural interactions between flagellin and TLR-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pro-inflammatory activity of flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flagellin and inflammatory disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adjuvant activity of flagellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adaptive immune response to flagellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bacterial flagellins have been studied for decades, due to the importance of these proteins in bacterial motility, and the complex nature of flagellar gene expression. However, renewed interest in flagellins arises from the recent understanding that these molecules are specifically recognized by ∗
Corresponding author. Tel.: +1 860 679 8785; fax: +1 860 679 1868. E-mail address: [email protected] (S.J. McSorley).
0165-2478/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2005.05.004
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the innate immune system. Therefore, these microbial products are members of a growing family of pathogen-associated molecule patterns (PAMPs), molecules that are used by the mammalian host to detect invasion by microbial pathogens. Recent work in our laboratory, and others, points to the fact that Salmonella flagellin is a target of both the innate and adaptive immune response during murine typhoid. Here, we discuss the interaction of bacterial flagellins with the innate and adaptive immune system, placing particular emphasis on the role of this interaction during Salmonella infection.
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2. Structure and function of flagellin Flagellin is the major protein constituent of bacterial flagella, complex surface appendages that are involved in bacterial locomotion. More than 50 genes are known to be involved in the regulated expression and function of the flagellum, implying that motility and chemotaxis are critically important for bacterial survival [1]. Flagella are part of the sensory machinery that allow bacteria to either respond to chemotactic stimuli, or simply avoid an unfavorable environment, such as extreme pH or saline concentrations. The structure is a self-assembling sub-system, consisting of a long helical filament emerging from a flexible hook, connected to a basal body that is anchored in the inner and outer cell membrane [1]. When in use, the flagellum exhibits a random pattern of movement characterized by two different turns, a counterclockwise rotation, when the bacteria packs all filaments into one structure, and an alternative clockwise rotation that causes separation of the filaments and chaotic movement of the bacteria [2]. Polymers of a single protein, flagellin, compose the 11 filaments of each flagellum. Salmonella flagellin is comprised of 494 amino acids and distinct domains have been described, based on homology between different Salmonella serovars [3–5]. Both the amino- and carboxy-terminus are well conserved among Salmonella serovars, while the central portion displays more diversity, one particular region being termed the “hypervariable region”. Recent analysis of the crystal structure of a Salmonella flagellin fragment revealed that the protein filament is folded back upon itself such that the amino- and carboxy-termini are physically located in close proximity to each other, within the central portion of the filament, and are likely involved in determining the repeating 3-D structure [6,7]. In contrast, the central portion of flagellin is exposed on the outside of the filament, providing a simple explanation for the fact that antibody responses tend to be targeted to this region [8].
3. The role of flagella in Salmonella pathogenesis Mucosal infection with bacterial strains that are genetically deficient in flagellin or flagellar-associated genes have demonstrated an obligatory role for the flagella in bacterial adhesion to epithelial surfaces, colonization, biofilm formation, and invasion of host tissues. For example, a flagellindeficient strain of Pseudomonas aeruginosa was found to be severely attenuated compared to the flagellated parental strain [9]. Histological analysis in this model demonstrated that flagellin-deficient strains cause a localized infection foci, in contrast to the typical spreading of infection observed during wild-type Pseudomonas pulmonary infection [9]. In another model system, expression of flagella is required for the persistence of Helicobacter pylori following infection of piglets [10]. For infection with Vibrio species, production of flagella is known to contribute to initial attachment of the bacteria
to the cell surface, biofilm formation and ultimate lethality [11,12]. Salmonella infection of inbred mouse strains is the best available laboratory model to study human typhoid fever [13] and flagellin expression is not normally considered to be a virulence factor [14,15]. However, recent experiments using rabbits uncovered a potential role for flagella in the initial interaction of Salmonella with M cells of the appendix [16]. Furthermore, flagellin expression is required for Salmonella invasiveness in a cell culture model, and for the induction of polymorphonuclear leukocyte infiltration using the calf intestine model of infection [15]. Therefore, although flagellin appears to be dispensable for Salmonella virulence in the mouse model, there are multiple lines of evidence to suggest that it is critically important for establishment of Salmonella infection in other species. The importance of flagellin expression to human typhoid fever remains to be established, but the available data from the models above suggest that it could play an important role in Salmonella typhi pathogenesis.
4. Structural interactions between flagellin and TLR-5 TLR-5 is expressed by epithelial cells, monocytes, and immature dendritic cells [17,18]. Initial identification of flagellin as the ligand for TLR-5 came from a study that isolated stimulatory components from Listeria culture supernatant proteins by HPLC [19]. Parallel studies examining the activation of epithelial cells in response to Salmonella also identified flagellin as a stimulatory ligand for TLR-5 [20]. Thus, TLR-5 can recognize flagellin that is produced by both Gram-positive and Gram-negative bacteria, providing a simple strategy for the host to respond to flagellated bacteria. In contrast to many other TLR ligands, the proteinaceous nature of flagellin has enabled a detailed study of the structural basis of flagellin–TLR-5 interactions. Two different groups have documented bioactivity of flagellin even after production in a eukaryotic expression system, demonstrating that the stimulatory capacity of flagellin is independent of other bacterial proteins or prokaryotic post-translational modifications [21,22]. Using defined deletion mutants of flagellin, two different groups initially identified two distinct sites required for bioactivity, the hypervariable region localized in domain 3, and the conserved amino- and carboxy-termini of the protein [23,24]. This discrepancy was resolved by more detailed mapping, which defined TLR-5 stimulatory activity within N-amino terminal residues 79–117 and 408–439 [22]. Thus, TLR-5 most likely recognizes a spatial area of flagellin comprising areas of the amino- and carboxy-terminus. The observation that the TLR-5 binding site of flagellin is most likely located within the central base of the filament is interesting since the residues needed for TLR-5 recognition are the same residues essential for the native structure of the flagellum and bacterial motility. Thus, the bacteria are unable to avoid host recognition by flagellin sequence muta-
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Fig. 1. Unresolved issues with regard to flagellin interactions with the immune system. Flagellin induces pro-inflammatory cytokine production and dendritic cell (DC) activation, eventually leading to CD4 T cell activation. However, several key aspects of this activity are unclear and are highlighted in the figure numerically. (1) What is the bioactive form of flagellin (polymer or oligomers) that activates pro-inflammatory activity? (2) Does flagellin induce DC maturation directly, via TLR-5 expressed on the DC, or (3) indirectly through cytokines produced by other cell types? (4) What type of adaptive immune response is induced by the adjuvant effect of flagellin, TH1, or TH2?
tion without compromising the beneficial aspects of bacterial motility. However, it is not yet clear how the internal structure of the flagellum filament would actually come in contact with TLR-5 in order to mediate pro-inflammatory activity in vivo (Fig. 1)? One possibility is that monomers or oligomers of flagellin are released from the filament in the hostile environment of the phagosome, or may be exposed during bacterial replication [25,26].
5. Pro-inflammatory activity of flagellin Initial studies using human blood mononuclear cells demonstrated that flagellin from different bacterial species stimulated the production of cytokines, such as TNF-␣ and IL-1- [25,27]. Furthermore, flagellin produced by S. typhi was found to induce synthesis of IL-6 and the antiinflammatory cytokine IL-10 [25,27–29]. More recent studies by Gewirtz et al. identified flagellin as the bacterial factor inducing IL-8 production following the interaction of Salmonella with human intestinal epithelial cells [30]. Interestingly, the activation of this inflammatory response was dependent upon the translocation of flagellin to the basolateral surface of the epithelial cells, where TLR5 is expressed [20,30]. Presumably, this requirement limits epithelial cell activation to pathogens that can translocate flagellin, and therefore, avoids TLR-5 ligation by the normal intestinal flora. Other studies have examined flagellin activation of human intestinal epithelial cell lines and noted the production of the chemokine CCL-20, and the subsequent migration of immature human DC in response to this stimu-
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lus [31]. In contrast to other studies, this report suggested that apical expression of TLR-5 was responsible for chemokine secretion [31]. In agreement with this notion, constitutive expression of TLR-5 has been reported on the apical surface of human primary IECs [17], and another recent study demonstrated that flagellin from commensal bacteria can induce TLR-5 activation on the apical surface of freshly isolated murine IECs [32]. Future studies are obviously required to resolve the issue of TLR-5 expression on the surface of epithelial cells and flagellin translocation (Fig. 1). If TLR-5 is expressed on the apical surface of epithelial cells, it remains unclear why a permanent inflammatory state is not induced in the intestinal mucosa in response to flagellated commensal bacterial. One possibility is that commensal microbes may also induce anti-inflammatory mediators or block inflammatory signaling pathways of epithelial cells [33,34]. TLR-5 is also highly expressed in the lung [35] and seems to play an important role in the defense against pathogens of the respiratory tract [36,37]. Interestingly, there is a correlation between a common human TLR-5 polymorphism and susceptibility to Legionnaires disease [38]. A singlepoint mutation to a stop codon at position 392 resulted in impaired responsiveness to flagellin in these patients [38]. Taken together, the available evidence suggests that expression of TLR-5 in the intestinal and respiratory mucosa is an important innate immune sensor for flagellated pathogens.
6. Flagellin and inflammatory disease Flagellin can induce expression of numerous proinflammatory mediators, such as TNF-␣, IL-1, IL-6, MIP-3␣, and iNOS, and therefore, may be involved in the development of bacterial-associated pathology. In support of this hypothesis, direct intratracheal instillation of 1 g of flagellin to mice resulted in an acute inflammatory process in the lung, involving neutrophil and macrophage infiltration and the detection of inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid [39]. Surprisingly, the administration of flagellin was more potent than LPS in the development of a lung inflammatory response [39], which could be due to low levels of TLR-4 in the lung, as described for intestinal epithelium [17]. It is possible, therefore, that chronic lung pathology caused by flagellated pathogens like Pseudomonas or Legionella is due in part to host responses to flagellin [37,38]. Analogous to endotoxin, flagellin is an effective mediator of systemic inflammation. Intravenous injection of mice with flagellin rapidly produced the typical pattern of cytokine, chemokine, and NO production, as well causing the clinic signs of septic shock; hypotension, respiratory distress, cyanosis, organ injury, and death [26,40]. Although it is not clear whether LPS or flagellin is more effective at causing toxic shock in mice [26,40,41], these data demonstrate that flagellin could be an important contributor to the inflammatory processes during bacterial sepsis.
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Crohn’s disease and ulcerative colitis are intestinal inflammatory disorders mediated by an abnormal immune response to commensal microbes. The identification of potential target antigens of the immune response has been a particular focus of research in this field. Recently, two different groups identified bacterial flagellin as an immunodominant antigen in Crohn’s disease [42,43]. High titers of anti-flagellin antibodies were detected in Crohn’s patients and colitic mice. Although these studies identified flagellin as a target of the adaptive immune response, it is also likely that the innate pro-inflammatory activity of bacterial flagellins can contribute to the pathology associated with this intestinal disease.
7. Adjuvant activity of flagellin As with other PAMPs, the function of flagellin as an adjuvant has been examined in detail. An initial report by our laboratory demonstrated that Salmonella flagellin was capable of enhancing antigen specific CD4+ T cell expansion and memory development in vivo [21]. This adjuvant effect of flagellin was dependent upon the ability to activate CD80/86 molecules expression and was completely blocked by administration of CTLA-4-Ig. Another interesting report noted that bacterial flagellin can directly activate human but not murine dendritic cells in vitro, suggesting that any adjuvant effect of flagellin in mice may be indirect and involve TLR-5 ligation on other cell types [44]. However, this latter observation has been challenged by another report that described direct flagellin activation of murine dendritic cells in vitro [45]. Differences in dendritic cell maturation or flagellin purification may explain these discrepancies between these studies. Preliminary data in our laboratory suggest that flagellin does not directly activate murine dendritic cells (Salazar and McSorley, unpublished data), and future experiments will hopefully resolve this important issue (Fig. 1). In our hands, and others, flagellin induced the development of antigen-specific Th1 response involving the production of IFN-␥ and not IL-4 upon recall stimulation [21,46,47]. These observations are consistent with a role for flagellin in contributing to the normal development of Th1 during murine Salmonella infection [48,49]. However, another report using flagellin as an adjuvant has described the generation of a Th2 CD4+ T cell response, finding IL-4, IL-13, and a typical Th2 antibody response after immunization [45]. This ability of flagellin to generate a Th2 response in vivo was corroborated by other group, who demonstrated convincingly that administration of soluble flagellin caused a Th2-like antibody response [50]. It remains unclear why flagellin can evoke such different T cell responses in different adjuvant model systems (Fig. 1). It seems likely that the purity of flagellin preparation, extent of polymerization, dose administered, and/or route of administration will explain these differences.
8. Adaptive immune response to flagellin As already noted above, bacterial flagellins are targets of the adaptive immune response during Crohn’s disease, and recent reports also suggest that flagellins may be important T and B cell targets during bacterial infection. Indeed, bacterial flagellins have been used for decades as target antigens to study the immune response in vivo. Elegant studies examining tolerance induction in rats and B cell antigen receptor recognition, made use of bacterial flagellin as a target antigen [51–53]. Furthermore, the polymeric structure and abundance of protein expressed by bacteria led to the use of flagellin as a carrier protein for B and T cell epitopes [54,55]. More recently, flagellin has been identified as a major target antigen of CD4+ T cell cells during murine and human Salmonella infection [56–58]. The poor definition of other targets of the adaptive immune response to Salmonella make evaluation of the importance of the flagellin-specific response difficult [59]. However, we do know that T cell responses to Salmonella flagellin occur rapidly after oral infection, and may contribute to vaccine-induced immunity [58,60]. Therefore, it seems likely that adaptive immune responses to flagellin are important in defense against bacterial infection.
9. Conclusion Bacterial flagellins are interesting microbial proteins that are targeted by both the innate and adaptive immune response. It seems likely that these proteins are critical to the development of protective immunity to bacterial infection, and the development of inflammatory conditions due to bacterial infection. The use of flagellins as part of vaccine or adjuvant preparations may lead to the development of novel live or sub-unit prophylactic treatments for several infectious diseases.
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