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Antigen-presenting cells and anti-Salmonella immunity
Microbes and Infection, 3, 2001, 1239−1248 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457901014848/REV
Antigen-presenting cells and anti-Salmonella immunity Ulf Yrlid, Mattias Svensson, Alun Kirby, Mary Jo Wick* Department of Cell and Molecular Biology, Section for Immunology, Lund University, BMC I-13, 221 84 Lund, Sweden
ABSTRACT – The present article summarizes studies aimed at addressing the role of antigenpresenting cell populations, particularly dendritic cells (DC), in the immune response to Salmonella typhimurium. Data from in vitro studies shed light on presentation of antigens expressed in Salmonella on major histocompatibility complex class I and class II molecules by infected DC and macrophages, and the activation state of DC following infection. Finally, data from in vivo studies addressing the role of DC and defined DC subsets during the host response to Salmonella using a murine infection model are discussed. © 2001 Éditions scientifiques et médicales Elsevier SAS Salmonella typhimurium / dendritic cell / macrophage / apoptosis / antigen presentation
1. Triggering antibacterial immunity Two types of phagocytic cells, macrophages and immature dendritic cells (DC), are particularly important in the interface between the innate and adaptive immune responses to bacterial infections. The sentinel function of these cells in capturing bacteria is coupled to their ability to act as antigen-presenting cells (APC). In this capacity, these phagocytic cells degrade bacteria and, in a wellorchestrated series of proteolytic events, process bacterial proteins into peptide fragments that are displayed on the APC surface by major histocompatibility complex (MHC) molecules [1]. Processing of exogenous antigens such as internalized bacteria typically generates peptides that bind MHC-II molecules. However, bacteria as well as other exogenous antigens can also be processed for peptide presentation on MHC-I molecules [2, 3]. Peptides presented by the two different classes of ’classical’ MHC molecules, MHC-I and MHC-II, are recognized by CD8+ and CD4+ T cells, respectively. Peptide–MHC-II complexes are recognized by specific CD4+ T cells which, in turn, provide the help necessary for B-cell activation and production of isotype-switched antibodies. Peptide–MHC-I complexes, on the other hand, are recognized by specific CD8+ cytotoxic T cells that destroy the cells expressing the complexes, such as cells infected with viruses, parasites or intracellular bacteria. Presentation of antigens from intracellular bacteria, which are initially extracellular upon entering a host but that can ultimately reside inside host cells, on both MHC-I and MHC-II is important in effective immunity to these pathogens. *Correspondence and reprints. E-mail address: [email protected] (M.J. Wick). Microbes and Infection 2001, 1239-0
2. Dendritic cells and macrophages: beauty and the beast Due to their harsh phagosomal milieu and extensive degradative capacity, macrophages have traditionally been considered the key APC in combating bacterial infections. However, the role of macrophages in stimulating naive T cells to initiate an immune response in vivo is unclear. This may be due to their insufficient surface expression of MHC and costimulatory molecules needed to trigger naive T cells and/or their marginal capacity to migrate from peripheral tissues, the sites of capturing bacterial invaders, to secondary lymphoid organs [4]. In contrast, mature DC efficiently trigger naive T cells and are critical for initiating primary immune responses [5]. DC reside in peripheral tissues in an immature state and can capture antigens including bacteria and parasites [5]. Immature DC, although very capable of capturing and processing antigens, are not yet efficient stimulators of T cells. This capacity is acquired in a process termed maturation. DC maturation is a coordinated process that restructures the biology of immature DC to make them effective activators of naive T cells [5, 6]. DC maturation includes downregulating antigen capture capacity, upregulating MHC and costimulatory molecule expression as well as altering chemokine production and chemokine receptor expression. Microbial products encountered in the periphery, such as LPS or nucleic acids, as well as proinflammatory cytokines such as TNF-α or IL-1β, trigger DC to mature [5, 6]. Thus, DC maturation induced by antigen and inflammatory stimuli optimizes their capacity to present antigens to and activate naive T cells in secondary lymphoid organs [5, 6]. 1239
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Although it is likely that both macrophages and DC contribute to antibacterial immunity, the precise role that these cells play may differ. Macrophages, for example, may function primarily to control bacterial replication and provide an initial source of cytokines such as IL-12 and TNF-α rather than initiating specific T-cell-dependent immunity. In contrast, DC may be the critical APC in triggering a T-cell response, particularly for stimulating naive T cells. DC activation of bacteria-specific T cells could occur by DC presentation of bacterial peptides to T cells in secondary lymphoid organs after antigen capture, either by direct phagocytic processing of bacteria or as bystander cells, concomitant with receipt of a maturation stimulus. To begin to dissect the role of phagocytic APC populations in antibacterial immunity, we have investigated various aspects of the APC function of both macrophages and DC using Salmonella typhimurium as a model.
3. S. typhimurium: where oh where art thou? As this Forum is dedicated to Salmonella, and diverse aspects of S. typhimurium pathogenesis have been superbly described elsewhere in the Forum, it is sufficient here to only briefly mention a few aspects of this bacterium relevant to the present chapter. S. typhimurium is an interesting model bacteria to study the processing and presentation of bacteria-derived peptides on MHC-I and MHC-II for several reasons. First, observations suggest that S. typhimurium can influence the ability of infected APC to present Salmonella-derived antigens to stimulate T cells. That is, this bacterium can survive and replicate in macrophages [7, 8] and DC [9], the very cells the host relies on to control replication and initiate specific immunity. Furthermore, S. typhimurium remains confined in, and alters the physiology of, phagocytic vacuoles of APC [10–12], endosomal compartments central to bacterial degradation and antigen processing. Finally, although S. typhimurium is a facultative intracellular bacterium confined to vacuolar compartments, cytotoxic T cells restricted to classical MHC-I molecules, as well as MHC class Ib molecules, are elicited following infection ([2, 13]; see note added in proof (1)). Thus, it is interesting to determine how antigens from S. typhimurium are presented on MHC-I, particularly as the major source of peptides presented on MHC-I is typically generated from endogenously synthesized, cytosolic proteins [14]. Understanding where and how Salmonella-derived peptides are generated for MHC-I presentation, as well as how virulence factors of this microbe influence antigen processing efficiency for MHC-I and MHC-II presentation, are important in understanding how efficient immunity to this microbe is generated. In addition, the involvement of macrophages versus DC in generating surface MHC molecules loaded with Salmonelladerived peptides in vivo and the mechanism used to generate these complexes, by direct presentation or as a bystander APC, are also important issues to address in anti-Salmonella immunity. 1240
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4. Direct presentation of Salmonella antigens by macrophages and DC Murine macrophages from different sources, including peritoneal-elicited and bone marrow-derived, process S. typhimurium for peptide presentation on MHC-I as well as MHC-II [2, 15–18]. The pathway used for MHC-I presentation of Salmonella antigens by macrophages does not require components of the cytosolic MHC-I presentation pathway. For example, Kb-restricted presentation of OVA(257–264) processed from Salmonella expressing the cytosolic Crl-OVA fusion protein, which contains amino acids 257–277 from ovalbumin (OVA) fused to the bacterial regulatory protein Crl, occurs in the absence of a functional transporter associated with antigen processing (TAP) [18]. TAP is a heterodimeric ER membrane protein that translocates peptides from the cytosol into the ER that is required for wild-type levels of surface MHC-I expression [19]. As TAP-deficient cells have a reduced pool of peptide-receptive post-Golgi and surface MHC-I molecules [19], the role of TAP in MHC-I presentation of antigens from phagocytosed bacteria may be indirect. Thus, the observed TAP-independent presentation of Salmonella-encoded antigens by macrophages is consistent with a mechanism where post-Golgi MHC-I molecules are used, and additional data supports this suggestion. For instance, MHC-I presentation of antigens expressed in Salmonella occurs in the presence of Brefeldin A, a compound which inhibits egress of newly synthesized proteins including MHC-I molecules from the ER to the Golgi, and in the presence of compounds that inhibit proteolytic activity of proteasomes [20]. It has also been shown that S. typhimurium-infected macrophages regurgitate bacteria-derived peptides that are loaded onto preformed surface MHC-I of pre-fixed bystander macrophages [21]. Taken together, these data suggest that alternate mechanism(s) including endosomal processing following bacterial internalization and use of post-Golgi MHC-I molecules are involved in MHC-I presentation of Salmonelladerived antigens from infected macrophages. However, the available data do not allow the exact pool of MHC-I molecules used in phagocytic antigen presentation by infected macrophages to be established. The peptide regurgitation experiments support a role for loading of surface MHC-I, but whether surface molecules are used alone or in combination with MHC-I in phagosomal and/or endosomal compartments in direct presentation of antigens from phagocytosed bacteria remains to be determined. An additional aspect of this presentation that is not clear is the following: if MHC-I molecules located in endosomal compartments of macrophages are used for presentation of bacteria-derived antigens, what is the means by which these MHC-I molecules are localized to endocytic vacuoles? For example, did they arrive in the endosomal system from the protein secretion apparatus or via recycling of molecules from the surface? [22]. If the former is the case, is the trafficking of MHC-I into the endosomal system mediated by invariant chain [23] or by another mechanism? Numerous studies report the presence of MHC-I in endosomal compartments of various cell types. However, Microbes and Infection 2001, 1239-0
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Figure 1. MHC-I presentation of model antigens expressed in S. typhimurium by bone marrow-derived DC. DC cultured from the bone marrow of either C57BL/6 or TAP1–/– mice were co-cultured with S. typhimurium χ4550 or with χ4550 expressing full-length OVA as indicated at a bacteria to DC ratio of 50:1 (A) or with 10 µM OVA(257-264) peptide (B) for 2 h. Where indicated, DC were pretreated with 10 µg/mL of cytochalasin D (+CCD) to inhibit cytoskeletal rearrangements and thus bacterial internalization. Following bacterial or peptide incubation, the DC were washed and incubated for an additional 2 h before fixing in paraformaldehyde. IL-2 production by CD8OVA T hybridoma cells, which secrete IL-2 upon specific recognition of OVA(257-264)/Kb, was then quantitated [24, 27]. DC derived from the bone marrow of either C57BL/6 or TAP1–/– mice were co-cultured with heat-killed (65 °C for 45 min) S. typhimurium 14028 expressing Crl-OVA (C) or with 1 nM OVA(257-264) peptide (D) for 2 h. The cultures were then washed and fresh media and CD8OVA T hybridoma cells were added. The incubation continued overnight and IL-2 in the culture supernatant was then quantitated. For all panels, the y axis represents the CD8OVA T hybridoma response.
the origin and contribution of these molecules to MHC-I presentation of Salmonella antigens by macrophages remains to be clarified. DC at an immature stage of differentiation also internalize and process S. typhimurium for presentation of bacteria-derived antigens on both MHC-I and MHC-II molecules [9, 24, 25]. Similarly to the case for macrophages, the mechanism used by DC to process Salmonella expressing Crl-OVA (Svensson and Wick, unpublished data) or full-length OVA (figure 1A) for OVA(257–264)/Kb presentation requires active internalization of the bacteria. However, unlike macrophages, DC presentation of Salmonella-derived peptides on MHC-I requires components of the classical (cytosolic) MHC-I presentation pathway. For example, Kb-restricted presentation of OVA(257–264) processed from S. typhimurium expressing Crl-OVA [26] or full-length OVA (figure 1A) is abolished in DC derived from TAP1–/– mice. Furthermore, TAP-dependent presentation of the Kb-restricted OVA epitope derived from Crl-OVA is observed when DC process either live [26] or heat-killed S. typhimurium (figure 1C). This demonstrates that an active process of live bacteria is not required for the Salmonella-encoded antigen to access the cytosol of an infected DC. Interestingly, unlike macrophages, DC that have phagocytosed Escherichia Microbes and Infection 2001, 1239-0
coli expressing Crl-OVA do not regurgitate bacteriaderived peptides for loading onto surface MHC-I of prefixed bystander cells [27]. Although a lack of regurgitation of Salmonella-derived peptides remains to be directly demonstrated, the data from experiments using E. coli suggest that peptide regurgitation and loading of preformed surface MHC-I molecules is not a mechanism used by DC to present peptides processed from internalized bacteria. Together these data suggest that, in contrast to the mechanism used by macrophages, DC presentation of Salmonella-encoded antigens on MHC-I uses the cytosolic presentation pathway. Indeed, DC may have a unique ability to transfer internalized antigen into the cytosol, as has been demonstrated for antigens internalized by Fc or mannose receptors [28]. However, such a mechanism remains to be demonstrated for antigens encoded in bacteria that cannot themselves escape into the cell cytosol.
5. Indirect presentation of Salmonella antigens by bystander cells It has been known for some time that S. typhimurium infection of cultured macrophages can be cytolytic [29, 30]. Both apoptotic as well as nonapoptotic (necrotic) 1241
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Figure 2. Logarithmically growing wild-type S. typhimurium strain 14028 induces apoptosis in DC while the phoPc mutant strain CS022 does not. Bone marrow-derived DC were co-incubated with increasing bacteria to DC ratios as indicated for 90 min. The cells were stained with Annexin V-FITC and propidium iodide and were analyzed by flow cytometry [25]. Histograms show gated propidium iodide negative cells plotted against cell number for DC co-incubated with either S. typhimurium 14028 (thick line), S. typhimurium CS022 (dotted line) or in medium only (thin line).
mechanisms appear to be associated with Salmonellainduced cell death [25, 29–33]. Infecting cultured bone marrow-derived macrophages [25] or DC (figure 2) with S. typhimurium expressing the type III secretion system clearly results in a population of cells exhibiting features of early apoptosis (i.e. staining positive with Annexin V while excluding propidium iodide). As a result of bacteriainduced cell death, direct presentation of Salmonelladerived antigens on both MHC-I and MHC-II by the infected cells is abrogated [25]. This observation, combined with the release of bacteria from dead cells that could subsequently infect and replicate in neighboring cells, may be a mechanism whereby Salmonella evades immune recognition and propagates itself in an infected host. However, Salmonella-induced apoptosis of macrophages provides a reservoir of Salmonella antigens that can be presented by bystander DC [25]. That is, neighboring DC ingest apoptotic material from macrophages induced to undergo apoptosis by Salmonella infection and present peptides from Salmonella-encoded antigens on MHC-I and MHC-II [25]. Interestingly, although bystander macrophages also ingest Salmonella-induced apoptotic macrophages, Salmonella-derived peptides are not presented by bystander macrophages [25]. This may be due to enhanced degradative capacity of macrophages compared to DC such that antigenic epitopes are destroyed in the macrophages containing apoptotic material. Indeed, simultaneous addition of both bystander macrophages and bystander DC with macrophages that have been induced to undergo apoptosis by Salmonella infection reduces the level of presentation of Salmonella-encoded antigens on MHC-I and MHC-II by the bystander DC (Yrlid and Wick, unpublished data). This suggests that macrophages compete for apoptotic material, thus limiting the availability of antigens for presentation by the bystander DC. In addition, macrophages and DC use different integrin receptors to internalize apoptotic material [34], thus the fate of ingested apoptotic cells may differ in the two cell types. Alternatively, the bystander macrophages could produce a soluble factor that acts to inhibit the ability of bystander DC to present apoptotic material or that has an inhibitory effect on the antigen-specific T cells [35]. 1242
What is the nature of the material released by macrophages, induced to undergo apoptosis by Salmonella infection, that accounts for the presentation by bystander DC? The cross-presenting material is not simply peptides released from dying or dead macrophages that bind preformed surface MHC-I molecules on bystander DC. This was demonstrated in experiments showing that prefixed bystander DC could not cross-present antigenic material from neighboring macrophages induced to undergo apoptosis by Salmonella infection ([25] and figure 3). However, material capable of cross-presentation is present in the supernatant fraction collected from sequential centrifugation (250 g and 2 500 g) of Salmonella-induced apoptotic macrophage cultures (figure 3). These conditions remove bacteria, apoptotic macrophages and macrophage debris but not soluble proteins. It is important to note that residual bacteria remaining in the culture supernatant are not responsible for the observed crosspresentation [25]. Finally, the material present in the supernatant that is responsible for cross-presentation requires live bystander DC (figure 3). This suggests that internalization and processing of apoptotic material is required to generate the presented peptides. The precise nature of the material from Salmonella-induced apoptotic macrophages responsible for cross-presentation by bystander DC has not yet been elucidated. One possible candidate consistent with the data would be heat-shock protein(s) chaperoning antigenic material that accesses the MHC-I presentation pathway [36]. It is easy to envision that Salmonella-induced death of infected macrophages may not be an entirely tidy process containing only cells dying by apoptotic mechanisms, as mentioned above. This raises the question of whether the presentation of Salmonella-encoded antigens by bystander DC is limited to apoptotic death of the infected neighboring macrophages or if material from necrotic macrophages can also be presented by bystander DC. In experiments using higher Salmonella to macrophage infection ratios to increase the relative proportion of necrotic to apoptotic cells, reduced MHC-I presentation of a Salmonellaencoded antigen by bystander DC was apparent (Yrlid and Wick, unpublished data). MHC-II presentation of Microbes and Infection 2001, 1239-0
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6. Salmonella and DC maturation
Figure 3. Antigenic material is contained both in the supernatant and the pellet fractions of bacteria-induced apoptotic macrophages, but does not contain free peptide. Bone marrow-derived macrophages (H-2d) were co-incubated with logarithmically growing wild-type S. typhimurium 14028r, which induces apoptosis in infected macrophages, or with the phoPc mutant strain CS022r, which does not induce apoptosis in infected macrophages [25] at a bacteria to macrophage infection ratio of 15:1 for 90 min as indicated. Both strains expressed the Crl-OVA fusion protein. Following washing and addition of gentamycin, the macrophages were incubated overnight. The media from the co-cultures were then centrifuged at 250 g and the pellet and supernatant fractions were collected. Half of the collected supernatant was then centrifuged a second time at 2 500 g. The supernatant from this centrifugation (250 g + 2 500 g; filled bars), the supernatant from the initial 250 g centrifugation (open bars) and the pellet from the initial 250 g centrifugation (striped bars) were then added to live or paraformaldehyde fixed DC of the correct haplotype (H-2b) to be recognized by CD8OVA T hybridoma cells. Antigenic material (OVA(257-264) processed from the bacteria expressing Crl-OVA used to infect the macrophages) contained in three different fractions and presented by the added H-2b bystander DC was then quantitated using CD8OVA T hybridoma cells. macrophage-derived material was also reduced, albeit to a lesser degree than that observed for MHC-I presentation. These data suggest that apoptotic death is not an absolute requirement for presentation of cell debris by bystander cells, particularly for MHC-II presentation [37]. MHC-I presentation of material from dying cells by bystander cells, however, may be more dependent on apoptotic material. This could reflect differences in the ability of apoptotic versus necrotic cellular material to access the exogenous MHC-I versus MHC-II antigen presentation pathways. Several genera of enteric pathogens, including Salmonella and Shigella, induce apoptosis in vitro and in vivo. This raises the question of the role of bacteria-induced cell death in the immune response to these bacteria. The role of bacteria-mediated activation of caspase-1, a mechanism by which Salmonella and Shigella induce apoptosis that is also critical for activation of the proinflammatory cytokines IL-1β and IL-18, in the innate response to these pathogens has recently been reported [38, 39]. The role of apoptosis induction on the adaptive immune response to these bacteria, however, remains to be determined. Microbes and Infection 2001, 1239-0
As discussed above, immature DC can present Salmonella-encoded antigens on MHC-I and MHC-II either directly or as bystander cells. Salmonella co-culture with immature DC not only results in presentation of Salmonella antigens, but also in DC maturation [9]. Surface expression of MHC-I, MHC-II, CD40, CD54, CD80 and CD86 are increased on immature DC following a brief co-incubation with Salmonella. With the exception of CD80, the effect of surface molecule expression is independent of bacterial viability or internalization. This observation suggests that bacterial LPS is largely responsible for the alteration in surface molecule expression. Indeed, co-culture of DC with LPS purified from S. typhimurium also results in upregulation of surface molecule expression [9]. Another important feature of DC induced to undergo maturation by co-culture with Salmonella or Salmonella LPS is that it downregulates the ability of DC to internalize and process subsequently encountered bacteria for peptide presentation on both MHC-I and MHC-II [9]. Such a feature may help focus the immune response during bacterial infection. Thus, DC encounter with S. typhimurium in peripheral tissues results in processing of bacteria for presentation of bacterial antigens on MHC-I and MHC-II. This is coupled to increased expression of surface molecules important in signaling the immune system and DC migration to secondary lymphoid organs. Thus, the net result is Salmonella antigens are displayed as surface peptide–MHC complexes on mature DC relocated to secondary lymphoid organs. As these DC have increased costimulatory capacity as a result of their encounter with Salmonella, they are optimally situated to stimulate naive T cells and initiate an immune response against this bacteria.
7. DC and anti-Salmonella immunity Features of DC including their ability to present bacteriaderived antigens as well as the induction of maturation following DC encounter with bacteria or bacterial products raises the issue of the role of DC during the in vivo response to Salmonella infection. In murine models, it has recently been shown that Salmonella is contained within DC of Peyer’s patches following oral infection [40] as well as within CD11c+MHC-II+ splenic DC following i.v. or i.p. administration (see note added in proof (1)). DC are also activated following S. typhimurium infection, as demonstrated by increased surface expression of CD40 and CD86 on CD11c+MHC-II+ splenocytes examined directly ex vivo as early as 7 days post infection (see note added in proof (1)). Finally, DC loaded with either heat-killed or viable S. typhimurium can prime both CD4+ and CD8+ Salmonella-specific T cells upon transfer into naive mice (see note added in proof (1)). These data suggest that DC are key antigen-presenting cells that prime naive T cells during Salmonella infection. In recent years, DC subpopulations that differ with respect to phenotype, function and localization in secondary lymphoid organs have been described [41]. In mice these subsets have been distinguished based on differen 1243
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tial expression of the CD8αα homodimer, resulting in the classification of CD11c+MHC-II+ DC into CD8α– and CD8α+ subsets. DC within the CD8α– subset also differ with respect to expression of CD4, generating additional populations characterized as CD8α–CD4+ and CD8α–CD4– DC [41]. Functional differences, including T-cell stimulatory capacity, cytokine production, ability to internalize antigens, and differential ability to direct the development of Th1 versus Th2 CD4+ T lymphocytes, have been attributed to CD8α– and CD8α+ subsets [41]. In addition, CD8α+ DC reside primarily in the T-cell areas of secondary lymphoid organs while CD8α– DC reside in areas of antigen entry into lymphoid organs, such as the splenic marginal zone and subepithelial dome of Peyer’s patches [41, 42]. To investigate the role of DC subsets during Salmonella infection, studies were performed to characterize quantitative and qualitative changes in defined DC subsets after oral Salmonella challenge (see note added in proof (2)). These studies revealed that S. typhimurium-infected mice have distinct alterations in the localization, number and function of the CD8α+, CD8α–CD4+ and CD8α–CD4– DC subsets in the spleen. For example, in situ studies showed that 5 days following oral infection, CD4+ DC present at the border of splenic B-cell follicles in naive or E. colichallenged mice were no longer present in mice that received Salmonella. CD4+ DC remained present, however, within the B-cell follicles of Salmonella-challenged mice. No overall quantitative change in the CD4+ DC subset was apparent, suggesting that a redistribution of this DC subset occurred in response to Salmonella infection. A significant influx of both CD8α+ and CD8α–CD4– DC in splenic red pulp in response to Salmonella was also apparent in situ, and this was reflected in a quantitative increase in both of these populations (see note added in proof (2)). TNF-α was also produced by the DC subsets, particularly amongst the CD8α+ population, following S. typhimurium infection. In situ, however, cells staining positive for TNF-α were coincident with GR1+ neutrophils in the red pulp rather than with CD11c+ DC. This suggests that TNF-α production by DC may be involved in aspects of the anti-Salmonella response other than controlling initial bacterial growth, which is likely done primarily by neutrophils and macrophages. This is supported by in situ studies showing that the latter cell types are found in clusters in the red pulp, that is, in granulomas containing Salmonella. As TNF-α is a proinflammatory cytokine capable of inducing DC maturation [5, 6], TNF-α production by DC following S. typhimurium infection may be involved in orchestrating DC maturation and migration in the spleen to link the innate and adaptive responses to this microbe. This may be mediated by alteration of chemokine receptor expression and chemokine production that directs DC into different microenvironments of lymphoid organs [42]. Although the significance of the observed subsetspecific changes in DC populations in response to S. typhimurium infection is not yet clear, these changes may, for example, be involved in differential skewing of the CD4+ T-cell response. This is particularly relevant in light of recent data showing that CD8α+ versus CD8α– DC differ1244
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entially direct the development of CD4+ T cells into cells producing a Th1 versus Th2 cytokine profile, respectively [41]. Understanding the relationship between the APC presenting Salmonella antigens and the influence on the ensuing T-cell response may thus provide insight into factors influencing the immune response to this intracellular pathogen.
8. Conclusions The data summarized here may suggest the following scenario in response to oral Salmonella infection (figure 4). CD11c+MHC-II+ DC, or more specifically CD8α– DC, which are abundant in the subepithelial dome of Peyer’s patches [42], may be the first cell to encounter and engulf orally administered bacteria [40] (figure 4A). Salmonella also reside inside splenic CD11c+MHC-II+ DC (see note added in proof (1)). Whether the appearance of Salmonella in splenic DC is due to transport of free bacteria via lymph to the thoracic duct and then via the blood to the spleen followed by internalization of bacteria by splenic resident DC is not yet known. If this is the case, the splenic DC subset(s) responsible for bacterial internalization and a functional difference correlating to bacterial internalization by the various DC subsets remains to be defined. Alternatively, Salmonella may be transported from the intestinal tract inside of cells, possibly inside CD11c+MHCII+ DC or via other cells included among CD18+ phagocytes [43]. Whether Salmonella penetration of the intestinal barrier is restricted to entry over Peyer’s patches, and if so, the relative contribution of M-cell entry versus penetration through or between Peyer’s patch epithelial cells remains to be clarified. Few CD11c-F4/80+ macrophages are present in the subepithelial dome of Peyer’s patches [42, 44]. This observation may explain the lack of evidence for a prominent role of these cells in internalizing bacteria in Peyer’s patches of mice given oral Salmonella [40]. However, CD11c–F4/80+ macrophages as well as CD11c+ DC are abundant in villi lamina propria [42]. This raises the issues of bacterial penetration through villus epithelial cells and the role of APC populations in the lamina propria in engulfing bacteria and/or transporting orally acquired Salmonella into deeper tissues (figure 4A). An important issue of anti-Salmonella immunity is where and by what APC population(s) are antigens from orally acquired Salmonella presented to prime specific T cells. CD4+ T cells specific for a Salmonella-encoded antigen are present in Peyer’s patches several weeks after oral bacterial administration [45]. However, it is not known if the antigen presentation and T-cell priming occurs in Peyer’s patches or elsewhere (i.e. mesenteric lymph nodes and/or spleen) and the cells subsequently home to Peyer’s patches. Furthermore, the relative contribution of direct presentation of Salmonella antigens by APC that engulf the bacteria versus indirect presentation by bystander cells that acquire debris from Salmonella-infected cells is also not yet established (figure 4A). This point raises the issue of whether there is a contribution of Salmonella-induced apoptosis and presentation by bystander cells in eliciting Microbes and Infection 2001, 1239-0
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Figure 4. Model of the possible role of DC during oral Salmonella infection. Panel A depicts a Peyer’s patch and adjacent villi with a B-cell follicle, germinal center (GC), intrafollicular regions (IFR) and draining lymphatics (L) indicated. Orally acquired Salmonella (green) may penetrate the intestinal epithelial barrier over the Peyer’s patch follicle or possibly via villus epithelium. CD11c+CD8α– DC (blue) in the subepithelial dome as well as CD11c+ DC (yellow) and CD11c–F4/80+ macrophages (brown) in the lamina propria are indicated [42]. CD11c+CD8α+ DC in the IFR region [42] are indicated by red cells, while T cells are depicted in blue. The villus to the right of the subepithelial dome depicts uptake of Salmonella by APC that do not necessarily undergo apoptosis. Such cells may exit the gut mucosa via draining lymphatics and access secondary lymphoid organs where they directly present Salmonella antigens to T cells. Alternatively, such APC may enter the IFR and directly present Salmonella antigens to T cells. The villus to the left of the dome suggests that bacteria penetrating the epithelium may induce apoptosis in lamina propria macrophages or DC indicated by the brown hatched and yellow hatched cells, respectively. Material from such dying cells may subsequently be picked up by bystander macrophages or DC (brown or yellow cells, respectively). The former cells may degrade the material while the latter may migrate into the IFR or to the draining lymph node to present Salmonella antigens as bystander cells. Arrows suggest possible movement of DC into the B- or T-cell areas and/or their exit from the Peyer’s patch via the draining lymphatics. Panel B shows a diagrammatic cross-section of the spleen with the splenic artery (SA), white pulp (WP) and red pulp (RP) indicated. The T-cell-rich periarterial lymphoid sheath (PALS) is indicated with a T and blue cells, while an adjacent B-cell follicle is indicated with a B and yellow cells. The location of other cells is indicated by gray, CD11c+CD8α–CD4– DC; red, CD11c+CD8α+ DC; brown, red pulp CD11c–F4/80+ macrophages, green, CD11c+CD4+ DC. Possible movement of these cell populations during Salmonella infection is indicated by arrows.
specific immunity to Salmonella. Salmonella penetrating the intestine over the subepithelial dome could induce death in the DC located there. Alternatively, bacteria entering via the villus epithelium could induce death in the DC or macrophages present in the villus lamina proMicrobes and Infection 2001, 1239-0
pria (figure 4A). Debris released from cells induced to die by Salmonella infection could subsequently be engulfed by neighboring bystander cells. In this sense, bystander macrophages may act to sequester Salmonella antigens from the immune system. In contrast, bystander DC that 1245
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engulf cellular debris could migrate to lymphoid organs [46] and, although not harboring the bacteria themselves, trigger Salmonella-specific T cells. Splenic DC subsets respond to oral Salmonella challenge, both with altered cytokine production and localization in splenic microenvironments (figure 4B (see note added in proof (2)). The function of CD4+ DC within the B-cell areas and those surrounding the follicles, as well as the mechanism underlying the redistribution of these cells, is presently not known. It is possible that DC located in the follicles could directly interact with B cells and influence the initiation and regulation of antibody production [47]. The significance of the increase in both CD8α+ and CD8α–CD4– splenic DC in the red pulp following oral S. typhimurium infection (see note added in proof (2)) also remains to be determined. The accumulation of these cells early during infection, as well as their production of TNF-α, may precede their maturation and redistribution into the T-cell areas. This is consistent with studies showing that DC redistribute to T-cell areas in mice injected with LPS or an antigenic parasite extract [48–50], observations suggesting that DC respond spatially to antigenic challenge to become poised to prime T cells. The mechanism underlying the redistribution of DC into T-cell areas as well as the host and bacterial factors that modulate DC redistribution in response to Salmonella infection are presently unknown. Finally, the influence of DC subsets presenting Salmonella antigens on the subsequent T-cell response is also an important issue to be addressed. Elucidating the role of DC, and the identified DC subsets, in the adaptive immune response to Salmonella will provide valuable insight into the role of antigen-presenting cell populations in bacterial immunity.
Acknowledgments Work from our laboratory is funded by grants from the Swedish Natural Sciences Research Council, the European Commission, the Swedish Medical Research Council, the foundations of Österlund, Kock, Wiberg, Kungliga Fysiografiska and Crafoord, the Swedish Society for Medical Research, and Lund University Medical Faculty. The authors are grateful to past and present members of our research group, colleagues in our department as well as colleagues at other institutions that we are fortunate to have fruitful and exciting interactions with.
. Note added in proof 1. Yrlid U., Svensson M., Håkansson A., Chambers B.J., Ljunggren H.-G., Wick M.J., In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection, Infect. Immun. 69 (2001) 5726–5735. 2. Kirby A.C., Yrlid U., Svensson M., Wick M.J., Differential involvement of dendritic cell subsets during acute Salmonella infection, J. Immunol. 166 (2001) 6802–6811. 1246
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[31] Hersh D., Monack D.M., Smith M.R., Ghori N., Falkow S., Zychlinsky A., The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1, Proc. Natl. Acad. Sci. USA 96 (1999) 2396–2401. [32] Watson P.R., Gautier A.V., Paulin S.M., Bland P., Jones P.W., Wallis T.S., Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis, Infect. Immun. 68 (2000) 3744–3747. [33] Jesenberger V., Procyk K.J., Yuan J., Reipert S., Baccarini M., Salmonella-induced caspase-2 activation in macrophages: a novel mechanism in pathogen-mediated apoptosis, J. Exp. Med. 192 (2000) 1035–1045. [34] Albert M.L., Pearce S.F.A., Francisco L.M., Sauter B., Roy P., Silverstein R.L., Bhardwaj N., Immature dendritic cells phagocytose apoptotic cells via αvb5 and CD36, and cross-present antigens to cytotoxic T lymphocytes, J. Exp. Med. 188 (1998) 1359–1368. [35] Savill J., Fadok V., Corpse clearance defines the meaning of cell death, Nature 407 (2000) 784–788. [36] Castellino R., Boucher P.E., Eichelberg K., Mayhew M., Rothman J.E., Houghton A.N., Germain R.N., Receptormediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways, J. Exp. Med. 191 (2000) 1957–1964. [37] Inaba K., Turley S., Yamaide F., Iyoda T., Mahnke K., Inaba M., Pack M., Subklewe M., Sauter B., Sheff D., Albert M., Bhardwaj N., Mellman I., Steinman R.M., Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products by dendritic cells, J. Exp. Med. 188 (1998) 2163–2173. [38] Sansonetti P.J., Phalipon A., Arondel J., Thirumalai K., Banerjee S., Akira S., Takeda K., Zychlinsky A., Caspase-1 activation of IL-1b and IL-18 are essential for Shigella flexneri-induced inflammation, Immunity 12 (2000) 581–590. [39] Monack D.M., Hersh D., Ghori N., Bouley D., Zychlinsky A., Falkow S., Salmonella exploits caspase-1 to colonize Peyer’s patches in a murine typhoid model, J. Exp. Med. 192 (2000) 249–258. [40] Hopkins S.A., Niedergang F., Corthesy-Theulaz I.E., Kraehenbuhl J.P., A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer’s patch dendritic cells, Cell. Microbiol. 2 (2000) 59–68. [41] Moser M., Murphy K.M., Dendritic cell regulation of TH1-TH2 development, Nat. Immunol. 1 (2000) 199–205. [42] Iwasaki A., Kelsall B.L., Localization of distinct Peyer’s patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3α, MIP-3b, and secondary lymphoid organ chemokine, J. Exp. Med. 191 (2000) 1381–1393. [43] Vasquez-Torres A., Jones-Carson J., Bäumler A.J., Falkow S., Valdivia R., Brown W., Le M., Berggren R., Parks W.T., Fang F.C., Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes, Nature 401 (1999) 804–808. [44] Kelsall B.L., Strober W., Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer’s patch, J. Exp. Med. 183 (1996) 237–247. 1247
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Microbes and Infection 2001, 1239-1248
Antigen-presenting cells and anti-Salmonella immunity Ulf Yrlid, Mattias Svensson, Alun Kirby, Mary Jo Wick* Department of Cell and Molecular Biology, Section for Immunology, Lund University, BMC I-13, 221 84 Lund, Sweden
ABSTRACT – The present article summarizes studies aimed at addressing the role of antigenpresenting cell populations, particularly dendritic cells (DC), in the immune response to Salmonella typhimurium. Data from in vitro studies shed light on presentation of antigens expressed in Salmonella on major histocompatibility complex class I and class II molecules by infected DC and macrophages, and the activation state of DC following infection. Finally, data from in vivo studies addressing the role of DC and defined DC subsets during the host response to Salmonella using a murine infection model are discussed. © 2001 Éditions scientifiques et médicales Elsevier SAS Salmonella typhimurium / dendritic cell / macrophage / apoptosis / antigen presentation
1. Triggering antibacterial immunity Two types of phagocytic cells, macrophages and immature dendritic cells (DC), are particularly important in the interface between the innate and adaptive immune responses to bacterial infections. The sentinel function of these cells in capturing bacteria is coupled to their ability to act as antigen-presenting cells (APC). In this capacity, these phagocytic cells degrade bacteria and, in a wellorchestrated series of proteolytic events, process bacterial proteins into peptide fragments that are displayed on the APC surface by major histocompatibility complex (MHC) molecules [1]. Processing of exogenous antigens such as internalized bacteria typically generates peptides that bind MHC-II molecules. However, bacteria as well as other exogenous antigens can also be processed for peptide presentation on MHC-I molecules [2, 3]. Peptides presented by the two different classes of ’classical’ MHC molecules, MHC-I and MHC-II, are recognized by CD8+ and CD4+ T cells, respectively. Peptide–MHC-II complexes are recognized by specific CD4+ T cells which, in turn, provide the help necessary for B-cell activation and production of isotype-switched antibodies. Peptide–MHC-I complexes, on the other hand, are recognized by specific CD8+ cytotoxic T cells that destroy the cells expressing the complexes, such as cells infected with viruses, parasites or intracellular bacteria. Presentation of antigens from intracellular bacteria, which are initially extracellular upon entering a host but that can ultimately reside inside host cells, on both MHC-I and MHC-II is important in effective immunity to these pathogens. *Correspondence and reprints. E-mail address: [email protected] (M.J. Wick). Microbes and Infection 2001, 1239-0
2. Dendritic cells and macrophages: beauty and the beast Due to their harsh phagosomal milieu and extensive degradative capacity, macrophages have traditionally been considered the key APC in combating bacterial infections. However, the role of macrophages in stimulating naive T cells to initiate an immune response in vivo is unclear. This may be due to their insufficient surface expression of MHC and costimulatory molecules needed to trigger naive T cells and/or their marginal capacity to migrate from peripheral tissues, the sites of capturing bacterial invaders, to secondary lymphoid organs [4]. In contrast, mature DC efficiently trigger naive T cells and are critical for initiating primary immune responses [5]. DC reside in peripheral tissues in an immature state and can capture antigens including bacteria and parasites [5]. Immature DC, although very capable of capturing and processing antigens, are not yet efficient stimulators of T cells. This capacity is acquired in a process termed maturation. DC maturation is a coordinated process that restructures the biology of immature DC to make them effective activators of naive T cells [5, 6]. DC maturation includes downregulating antigen capture capacity, upregulating MHC and costimulatory molecule expression as well as altering chemokine production and chemokine receptor expression. Microbial products encountered in the periphery, such as LPS or nucleic acids, as well as proinflammatory cytokines such as TNF-α or IL-1β, trigger DC to mature [5, 6]. Thus, DC maturation induced by antigen and inflammatory stimuli optimizes their capacity to present antigens to and activate naive T cells in secondary lymphoid organs [5, 6]. 1239
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Although it is likely that both macrophages and DC contribute to antibacterial immunity, the precise role that these cells play may differ. Macrophages, for example, may function primarily to control bacterial replication and provide an initial source of cytokines such as IL-12 and TNF-α rather than initiating specific T-cell-dependent immunity. In contrast, DC may be the critical APC in triggering a T-cell response, particularly for stimulating naive T cells. DC activation of bacteria-specific T cells could occur by DC presentation of bacterial peptides to T cells in secondary lymphoid organs after antigen capture, either by direct phagocytic processing of bacteria or as bystander cells, concomitant with receipt of a maturation stimulus. To begin to dissect the role of phagocytic APC populations in antibacterial immunity, we have investigated various aspects of the APC function of both macrophages and DC using Salmonella typhimurium as a model.
3. S. typhimurium: where oh where art thou? As this Forum is dedicated to Salmonella, and diverse aspects of S. typhimurium pathogenesis have been superbly described elsewhere in the Forum, it is sufficient here to only briefly mention a few aspects of this bacterium relevant to the present chapter. S. typhimurium is an interesting model bacteria to study the processing and presentation of bacteria-derived peptides on MHC-I and MHC-II for several reasons. First, observations suggest that S. typhimurium can influence the ability of infected APC to present Salmonella-derived antigens to stimulate T cells. That is, this bacterium can survive and replicate in macrophages [7, 8] and DC [9], the very cells the host relies on to control replication and initiate specific immunity. Furthermore, S. typhimurium remains confined in, and alters the physiology of, phagocytic vacuoles of APC [10–12], endosomal compartments central to bacterial degradation and antigen processing. Finally, although S. typhimurium is a facultative intracellular bacterium confined to vacuolar compartments, cytotoxic T cells restricted to classical MHC-I molecules, as well as MHC class Ib molecules, are elicited following infection ([2, 13]; see note added in proof (1)). Thus, it is interesting to determine how antigens from S. typhimurium are presented on MHC-I, particularly as the major source of peptides presented on MHC-I is typically generated from endogenously synthesized, cytosolic proteins [14]. Understanding where and how Salmonella-derived peptides are generated for MHC-I presentation, as well as how virulence factors of this microbe influence antigen processing efficiency for MHC-I and MHC-II presentation, are important in understanding how efficient immunity to this microbe is generated. In addition, the involvement of macrophages versus DC in generating surface MHC molecules loaded with Salmonelladerived peptides in vivo and the mechanism used to generate these complexes, by direct presentation or as a bystander APC, are also important issues to address in anti-Salmonella immunity. 1240
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4. Direct presentation of Salmonella antigens by macrophages and DC Murine macrophages from different sources, including peritoneal-elicited and bone marrow-derived, process S. typhimurium for peptide presentation on MHC-I as well as MHC-II [2, 15–18]. The pathway used for MHC-I presentation of Salmonella antigens by macrophages does not require components of the cytosolic MHC-I presentation pathway. For example, Kb-restricted presentation of OVA(257–264) processed from Salmonella expressing the cytosolic Crl-OVA fusion protein, which contains amino acids 257–277 from ovalbumin (OVA) fused to the bacterial regulatory protein Crl, occurs in the absence of a functional transporter associated with antigen processing (TAP) [18]. TAP is a heterodimeric ER membrane protein that translocates peptides from the cytosol into the ER that is required for wild-type levels of surface MHC-I expression [19]. As TAP-deficient cells have a reduced pool of peptide-receptive post-Golgi and surface MHC-I molecules [19], the role of TAP in MHC-I presentation of antigens from phagocytosed bacteria may be indirect. Thus, the observed TAP-independent presentation of Salmonella-encoded antigens by macrophages is consistent with a mechanism where post-Golgi MHC-I molecules are used, and additional data supports this suggestion. For instance, MHC-I presentation of antigens expressed in Salmonella occurs in the presence of Brefeldin A, a compound which inhibits egress of newly synthesized proteins including MHC-I molecules from the ER to the Golgi, and in the presence of compounds that inhibit proteolytic activity of proteasomes [20]. It has also been shown that S. typhimurium-infected macrophages regurgitate bacteria-derived peptides that are loaded onto preformed surface MHC-I of pre-fixed bystander macrophages [21]. Taken together, these data suggest that alternate mechanism(s) including endosomal processing following bacterial internalization and use of post-Golgi MHC-I molecules are involved in MHC-I presentation of Salmonelladerived antigens from infected macrophages. However, the available data do not allow the exact pool of MHC-I molecules used in phagocytic antigen presentation by infected macrophages to be established. The peptide regurgitation experiments support a role for loading of surface MHC-I, but whether surface molecules are used alone or in combination with MHC-I in phagosomal and/or endosomal compartments in direct presentation of antigens from phagocytosed bacteria remains to be determined. An additional aspect of this presentation that is not clear is the following: if MHC-I molecules located in endosomal compartments of macrophages are used for presentation of bacteria-derived antigens, what is the means by which these MHC-I molecules are localized to endocytic vacuoles? For example, did they arrive in the endosomal system from the protein secretion apparatus or via recycling of molecules from the surface? [22]. If the former is the case, is the trafficking of MHC-I into the endosomal system mediated by invariant chain [23] or by another mechanism? Numerous studies report the presence of MHC-I in endosomal compartments of various cell types. However, Microbes and Infection 2001, 1239-0
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Figure 1. MHC-I presentation of model antigens expressed in S. typhimurium by bone marrow-derived DC. DC cultured from the bone marrow of either C57BL/6 or TAP1–/– mice were co-cultured with S. typhimurium χ4550 or with χ4550 expressing full-length OVA as indicated at a bacteria to DC ratio of 50:1 (A) or with 10 µM OVA(257-264) peptide (B) for 2 h. Where indicated, DC were pretreated with 10 µg/mL of cytochalasin D (+CCD) to inhibit cytoskeletal rearrangements and thus bacterial internalization. Following bacterial or peptide incubation, the DC were washed and incubated for an additional 2 h before fixing in paraformaldehyde. IL-2 production by CD8OVA T hybridoma cells, which secrete IL-2 upon specific recognition of OVA(257-264)/Kb, was then quantitated [24, 27]. DC derived from the bone marrow of either C57BL/6 or TAP1–/– mice were co-cultured with heat-killed (65 °C for 45 min) S. typhimurium 14028 expressing Crl-OVA (C) or with 1 nM OVA(257-264) peptide (D) for 2 h. The cultures were then washed and fresh media and CD8OVA T hybridoma cells were added. The incubation continued overnight and IL-2 in the culture supernatant was then quantitated. For all panels, the y axis represents the CD8OVA T hybridoma response.
the origin and contribution of these molecules to MHC-I presentation of Salmonella antigens by macrophages remains to be clarified. DC at an immature stage of differentiation also internalize and process S. typhimurium for presentation of bacteria-derived antigens on both MHC-I and MHC-II molecules [9, 24, 25]. Similarly to the case for macrophages, the mechanism used by DC to process Salmonella expressing Crl-OVA (Svensson and Wick, unpublished data) or full-length OVA (figure 1A) for OVA(257–264)/Kb presentation requires active internalization of the bacteria. However, unlike macrophages, DC presentation of Salmonella-derived peptides on MHC-I requires components of the classical (cytosolic) MHC-I presentation pathway. For example, Kb-restricted presentation of OVA(257–264) processed from S. typhimurium expressing Crl-OVA [26] or full-length OVA (figure 1A) is abolished in DC derived from TAP1–/– mice. Furthermore, TAP-dependent presentation of the Kb-restricted OVA epitope derived from Crl-OVA is observed when DC process either live [26] or heat-killed S. typhimurium (figure 1C). This demonstrates that an active process of live bacteria is not required for the Salmonella-encoded antigen to access the cytosol of an infected DC. Interestingly, unlike macrophages, DC that have phagocytosed Escherichia Microbes and Infection 2001, 1239-0
coli expressing Crl-OVA do not regurgitate bacteriaderived peptides for loading onto surface MHC-I of prefixed bystander cells [27]. Although a lack of regurgitation of Salmonella-derived peptides remains to be directly demonstrated, the data from experiments using E. coli suggest that peptide regurgitation and loading of preformed surface MHC-I molecules is not a mechanism used by DC to present peptides processed from internalized bacteria. Together these data suggest that, in contrast to the mechanism used by macrophages, DC presentation of Salmonella-encoded antigens on MHC-I uses the cytosolic presentation pathway. Indeed, DC may have a unique ability to transfer internalized antigen into the cytosol, as has been demonstrated for antigens internalized by Fc or mannose receptors [28]. However, such a mechanism remains to be demonstrated for antigens encoded in bacteria that cannot themselves escape into the cell cytosol.
5. Indirect presentation of Salmonella antigens by bystander cells It has been known for some time that S. typhimurium infection of cultured macrophages can be cytolytic [29, 30]. Both apoptotic as well as nonapoptotic (necrotic) 1241
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Figure 2. Logarithmically growing wild-type S. typhimurium strain 14028 induces apoptosis in DC while the phoPc mutant strain CS022 does not. Bone marrow-derived DC were co-incubated with increasing bacteria to DC ratios as indicated for 90 min. The cells were stained with Annexin V-FITC and propidium iodide and were analyzed by flow cytometry [25]. Histograms show gated propidium iodide negative cells plotted against cell number for DC co-incubated with either S. typhimurium 14028 (thick line), S. typhimurium CS022 (dotted line) or in medium only (thin line).
mechanisms appear to be associated with Salmonellainduced cell death [25, 29–33]. Infecting cultured bone marrow-derived macrophages [25] or DC (figure 2) with S. typhimurium expressing the type III secretion system clearly results in a population of cells exhibiting features of early apoptosis (i.e. staining positive with Annexin V while excluding propidium iodide). As a result of bacteriainduced cell death, direct presentation of Salmonelladerived antigens on both MHC-I and MHC-II by the infected cells is abrogated [25]. This observation, combined with the release of bacteria from dead cells that could subsequently infect and replicate in neighboring cells, may be a mechanism whereby Salmonella evades immune recognition and propagates itself in an infected host. However, Salmonella-induced apoptosis of macrophages provides a reservoir of Salmonella antigens that can be presented by bystander DC [25]. That is, neighboring DC ingest apoptotic material from macrophages induced to undergo apoptosis by Salmonella infection and present peptides from Salmonella-encoded antigens on MHC-I and MHC-II [25]. Interestingly, although bystander macrophages also ingest Salmonella-induced apoptotic macrophages, Salmonella-derived peptides are not presented by bystander macrophages [25]. This may be due to enhanced degradative capacity of macrophages compared to DC such that antigenic epitopes are destroyed in the macrophages containing apoptotic material. Indeed, simultaneous addition of both bystander macrophages and bystander DC with macrophages that have been induced to undergo apoptosis by Salmonella infection reduces the level of presentation of Salmonella-encoded antigens on MHC-I and MHC-II by the bystander DC (Yrlid and Wick, unpublished data). This suggests that macrophages compete for apoptotic material, thus limiting the availability of antigens for presentation by the bystander DC. In addition, macrophages and DC use different integrin receptors to internalize apoptotic material [34], thus the fate of ingested apoptotic cells may differ in the two cell types. Alternatively, the bystander macrophages could produce a soluble factor that acts to inhibit the ability of bystander DC to present apoptotic material or that has an inhibitory effect on the antigen-specific T cells [35]. 1242
What is the nature of the material released by macrophages, induced to undergo apoptosis by Salmonella infection, that accounts for the presentation by bystander DC? The cross-presenting material is not simply peptides released from dying or dead macrophages that bind preformed surface MHC-I molecules on bystander DC. This was demonstrated in experiments showing that prefixed bystander DC could not cross-present antigenic material from neighboring macrophages induced to undergo apoptosis by Salmonella infection ([25] and figure 3). However, material capable of cross-presentation is present in the supernatant fraction collected from sequential centrifugation (250 g and 2 500 g) of Salmonella-induced apoptotic macrophage cultures (figure 3). These conditions remove bacteria, apoptotic macrophages and macrophage debris but not soluble proteins. It is important to note that residual bacteria remaining in the culture supernatant are not responsible for the observed crosspresentation [25]. Finally, the material present in the supernatant that is responsible for cross-presentation requires live bystander DC (figure 3). This suggests that internalization and processing of apoptotic material is required to generate the presented peptides. The precise nature of the material from Salmonella-induced apoptotic macrophages responsible for cross-presentation by bystander DC has not yet been elucidated. One possible candidate consistent with the data would be heat-shock protein(s) chaperoning antigenic material that accesses the MHC-I presentation pathway [36]. It is easy to envision that Salmonella-induced death of infected macrophages may not be an entirely tidy process containing only cells dying by apoptotic mechanisms, as mentioned above. This raises the question of whether the presentation of Salmonella-encoded antigens by bystander DC is limited to apoptotic death of the infected neighboring macrophages or if material from necrotic macrophages can also be presented by bystander DC. In experiments using higher Salmonella to macrophage infection ratios to increase the relative proportion of necrotic to apoptotic cells, reduced MHC-I presentation of a Salmonellaencoded antigen by bystander DC was apparent (Yrlid and Wick, unpublished data). MHC-II presentation of Microbes and Infection 2001, 1239-0
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6. Salmonella and DC maturation
Figure 3. Antigenic material is contained both in the supernatant and the pellet fractions of bacteria-induced apoptotic macrophages, but does not contain free peptide. Bone marrow-derived macrophages (H-2d) were co-incubated with logarithmically growing wild-type S. typhimurium 14028r, which induces apoptosis in infected macrophages, or with the phoPc mutant strain CS022r, which does not induce apoptosis in infected macrophages [25] at a bacteria to macrophage infection ratio of 15:1 for 90 min as indicated. Both strains expressed the Crl-OVA fusion protein. Following washing and addition of gentamycin, the macrophages were incubated overnight. The media from the co-cultures were then centrifuged at 250 g and the pellet and supernatant fractions were collected. Half of the collected supernatant was then centrifuged a second time at 2 500 g. The supernatant from this centrifugation (250 g + 2 500 g; filled bars), the supernatant from the initial 250 g centrifugation (open bars) and the pellet from the initial 250 g centrifugation (striped bars) were then added to live or paraformaldehyde fixed DC of the correct haplotype (H-2b) to be recognized by CD8OVA T hybridoma cells. Antigenic material (OVA(257-264) processed from the bacteria expressing Crl-OVA used to infect the macrophages) contained in three different fractions and presented by the added H-2b bystander DC was then quantitated using CD8OVA T hybridoma cells. macrophage-derived material was also reduced, albeit to a lesser degree than that observed for MHC-I presentation. These data suggest that apoptotic death is not an absolute requirement for presentation of cell debris by bystander cells, particularly for MHC-II presentation [37]. MHC-I presentation of material from dying cells by bystander cells, however, may be more dependent on apoptotic material. This could reflect differences in the ability of apoptotic versus necrotic cellular material to access the exogenous MHC-I versus MHC-II antigen presentation pathways. Several genera of enteric pathogens, including Salmonella and Shigella, induce apoptosis in vitro and in vivo. This raises the question of the role of bacteria-induced cell death in the immune response to these bacteria. The role of bacteria-mediated activation of caspase-1, a mechanism by which Salmonella and Shigella induce apoptosis that is also critical for activation of the proinflammatory cytokines IL-1β and IL-18, in the innate response to these pathogens has recently been reported [38, 39]. The role of apoptosis induction on the adaptive immune response to these bacteria, however, remains to be determined. Microbes and Infection 2001, 1239-0
As discussed above, immature DC can present Salmonella-encoded antigens on MHC-I and MHC-II either directly or as bystander cells. Salmonella co-culture with immature DC not only results in presentation of Salmonella antigens, but also in DC maturation [9]. Surface expression of MHC-I, MHC-II, CD40, CD54, CD80 and CD86 are increased on immature DC following a brief co-incubation with Salmonella. With the exception of CD80, the effect of surface molecule expression is independent of bacterial viability or internalization. This observation suggests that bacterial LPS is largely responsible for the alteration in surface molecule expression. Indeed, co-culture of DC with LPS purified from S. typhimurium also results in upregulation of surface molecule expression [9]. Another important feature of DC induced to undergo maturation by co-culture with Salmonella or Salmonella LPS is that it downregulates the ability of DC to internalize and process subsequently encountered bacteria for peptide presentation on both MHC-I and MHC-II [9]. Such a feature may help focus the immune response during bacterial infection. Thus, DC encounter with S. typhimurium in peripheral tissues results in processing of bacteria for presentation of bacterial antigens on MHC-I and MHC-II. This is coupled to increased expression of surface molecules important in signaling the immune system and DC migration to secondary lymphoid organs. Thus, the net result is Salmonella antigens are displayed as surface peptide–MHC complexes on mature DC relocated to secondary lymphoid organs. As these DC have increased costimulatory capacity as a result of their encounter with Salmonella, they are optimally situated to stimulate naive T cells and initiate an immune response against this bacteria.
7. DC and anti-Salmonella immunity Features of DC including their ability to present bacteriaderived antigens as well as the induction of maturation following DC encounter with bacteria or bacterial products raises the issue of the role of DC during the in vivo response to Salmonella infection. In murine models, it has recently been shown that Salmonella is contained within DC of Peyer’s patches following oral infection [40] as well as within CD11c+MHC-II+ splenic DC following i.v. or i.p. administration (see note added in proof (1)). DC are also activated following S. typhimurium infection, as demonstrated by increased surface expression of CD40 and CD86 on CD11c+MHC-II+ splenocytes examined directly ex vivo as early as 7 days post infection (see note added in proof (1)). Finally, DC loaded with either heat-killed or viable S. typhimurium can prime both CD4+ and CD8+ Salmonella-specific T cells upon transfer into naive mice (see note added in proof (1)). These data suggest that DC are key antigen-presenting cells that prime naive T cells during Salmonella infection. In recent years, DC subpopulations that differ with respect to phenotype, function and localization in secondary lymphoid organs have been described [41]. In mice these subsets have been distinguished based on differen 1243
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tial expression of the CD8αα homodimer, resulting in the classification of CD11c+MHC-II+ DC into CD8α– and CD8α+ subsets. DC within the CD8α– subset also differ with respect to expression of CD4, generating additional populations characterized as CD8α–CD4+ and CD8α–CD4– DC [41]. Functional differences, including T-cell stimulatory capacity, cytokine production, ability to internalize antigens, and differential ability to direct the development of Th1 versus Th2 CD4+ T lymphocytes, have been attributed to CD8α– and CD8α+ subsets [41]. In addition, CD8α+ DC reside primarily in the T-cell areas of secondary lymphoid organs while CD8α– DC reside in areas of antigen entry into lymphoid organs, such as the splenic marginal zone and subepithelial dome of Peyer’s patches [41, 42]. To investigate the role of DC subsets during Salmonella infection, studies were performed to characterize quantitative and qualitative changes in defined DC subsets after oral Salmonella challenge (see note added in proof (2)). These studies revealed that S. typhimurium-infected mice have distinct alterations in the localization, number and function of the CD8α+, CD8α–CD4+ and CD8α–CD4– DC subsets in the spleen. For example, in situ studies showed that 5 days following oral infection, CD4+ DC present at the border of splenic B-cell follicles in naive or E. colichallenged mice were no longer present in mice that received Salmonella. CD4+ DC remained present, however, within the B-cell follicles of Salmonella-challenged mice. No overall quantitative change in the CD4+ DC subset was apparent, suggesting that a redistribution of this DC subset occurred in response to Salmonella infection. A significant influx of both CD8α+ and CD8α–CD4– DC in splenic red pulp in response to Salmonella was also apparent in situ, and this was reflected in a quantitative increase in both of these populations (see note added in proof (2)). TNF-α was also produced by the DC subsets, particularly amongst the CD8α+ population, following S. typhimurium infection. In situ, however, cells staining positive for TNF-α were coincident with GR1+ neutrophils in the red pulp rather than with CD11c+ DC. This suggests that TNF-α production by DC may be involved in aspects of the anti-Salmonella response other than controlling initial bacterial growth, which is likely done primarily by neutrophils and macrophages. This is supported by in situ studies showing that the latter cell types are found in clusters in the red pulp, that is, in granulomas containing Salmonella. As TNF-α is a proinflammatory cytokine capable of inducing DC maturation [5, 6], TNF-α production by DC following S. typhimurium infection may be involved in orchestrating DC maturation and migration in the spleen to link the innate and adaptive responses to this microbe. This may be mediated by alteration of chemokine receptor expression and chemokine production that directs DC into different microenvironments of lymphoid organs [42]. Although the significance of the observed subsetspecific changes in DC populations in response to S. typhimurium infection is not yet clear, these changes may, for example, be involved in differential skewing of the CD4+ T-cell response. This is particularly relevant in light of recent data showing that CD8α+ versus CD8α– DC differ1244
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entially direct the development of CD4+ T cells into cells producing a Th1 versus Th2 cytokine profile, respectively [41]. Understanding the relationship between the APC presenting Salmonella antigens and the influence on the ensuing T-cell response may thus provide insight into factors influencing the immune response to this intracellular pathogen.
8. Conclusions The data summarized here may suggest the following scenario in response to oral Salmonella infection (figure 4). CD11c+MHC-II+ DC, or more specifically CD8α– DC, which are abundant in the subepithelial dome of Peyer’s patches [42], may be the first cell to encounter and engulf orally administered bacteria [40] (figure 4A). Salmonella also reside inside splenic CD11c+MHC-II+ DC (see note added in proof (1)). Whether the appearance of Salmonella in splenic DC is due to transport of free bacteria via lymph to the thoracic duct and then via the blood to the spleen followed by internalization of bacteria by splenic resident DC is not yet known. If this is the case, the splenic DC subset(s) responsible for bacterial internalization and a functional difference correlating to bacterial internalization by the various DC subsets remains to be defined. Alternatively, Salmonella may be transported from the intestinal tract inside of cells, possibly inside CD11c+MHCII+ DC or via other cells included among CD18+ phagocytes [43]. Whether Salmonella penetration of the intestinal barrier is restricted to entry over Peyer’s patches, and if so, the relative contribution of M-cell entry versus penetration through or between Peyer’s patch epithelial cells remains to be clarified. Few CD11c-F4/80+ macrophages are present in the subepithelial dome of Peyer’s patches [42, 44]. This observation may explain the lack of evidence for a prominent role of these cells in internalizing bacteria in Peyer’s patches of mice given oral Salmonella [40]. However, CD11c–F4/80+ macrophages as well as CD11c+ DC are abundant in villi lamina propria [42]. This raises the issues of bacterial penetration through villus epithelial cells and the role of APC populations in the lamina propria in engulfing bacteria and/or transporting orally acquired Salmonella into deeper tissues (figure 4A). An important issue of anti-Salmonella immunity is where and by what APC population(s) are antigens from orally acquired Salmonella presented to prime specific T cells. CD4+ T cells specific for a Salmonella-encoded antigen are present in Peyer’s patches several weeks after oral bacterial administration [45]. However, it is not known if the antigen presentation and T-cell priming occurs in Peyer’s patches or elsewhere (i.e. mesenteric lymph nodes and/or spleen) and the cells subsequently home to Peyer’s patches. Furthermore, the relative contribution of direct presentation of Salmonella antigens by APC that engulf the bacteria versus indirect presentation by bystander cells that acquire debris from Salmonella-infected cells is also not yet established (figure 4A). This point raises the issue of whether there is a contribution of Salmonella-induced apoptosis and presentation by bystander cells in eliciting Microbes and Infection 2001, 1239-0
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Figure 4. Model of the possible role of DC during oral Salmonella infection. Panel A depicts a Peyer’s patch and adjacent villi with a B-cell follicle, germinal center (GC), intrafollicular regions (IFR) and draining lymphatics (L) indicated. Orally acquired Salmonella (green) may penetrate the intestinal epithelial barrier over the Peyer’s patch follicle or possibly via villus epithelium. CD11c+CD8α– DC (blue) in the subepithelial dome as well as CD11c+ DC (yellow) and CD11c–F4/80+ macrophages (brown) in the lamina propria are indicated [42]. CD11c+CD8α+ DC in the IFR region [42] are indicated by red cells, while T cells are depicted in blue. The villus to the right of the subepithelial dome depicts uptake of Salmonella by APC that do not necessarily undergo apoptosis. Such cells may exit the gut mucosa via draining lymphatics and access secondary lymphoid organs where they directly present Salmonella antigens to T cells. Alternatively, such APC may enter the IFR and directly present Salmonella antigens to T cells. The villus to the left of the dome suggests that bacteria penetrating the epithelium may induce apoptosis in lamina propria macrophages or DC indicated by the brown hatched and yellow hatched cells, respectively. Material from such dying cells may subsequently be picked up by bystander macrophages or DC (brown or yellow cells, respectively). The former cells may degrade the material while the latter may migrate into the IFR or to the draining lymph node to present Salmonella antigens as bystander cells. Arrows suggest possible movement of DC into the B- or T-cell areas and/or their exit from the Peyer’s patch via the draining lymphatics. Panel B shows a diagrammatic cross-section of the spleen with the splenic artery (SA), white pulp (WP) and red pulp (RP) indicated. The T-cell-rich periarterial lymphoid sheath (PALS) is indicated with a T and blue cells, while an adjacent B-cell follicle is indicated with a B and yellow cells. The location of other cells is indicated by gray, CD11c+CD8α–CD4– DC; red, CD11c+CD8α+ DC; brown, red pulp CD11c–F4/80+ macrophages, green, CD11c+CD4+ DC. Possible movement of these cell populations during Salmonella infection is indicated by arrows.
specific immunity to Salmonella. Salmonella penetrating the intestine over the subepithelial dome could induce death in the DC located there. Alternatively, bacteria entering via the villus epithelium could induce death in the DC or macrophages present in the villus lamina proMicrobes and Infection 2001, 1239-0
pria (figure 4A). Debris released from cells induced to die by Salmonella infection could subsequently be engulfed by neighboring bystander cells. In this sense, bystander macrophages may act to sequester Salmonella antigens from the immune system. In contrast, bystander DC that 1245
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engulf cellular debris could migrate to lymphoid organs [46] and, although not harboring the bacteria themselves, trigger Salmonella-specific T cells. Splenic DC subsets respond to oral Salmonella challenge, both with altered cytokine production and localization in splenic microenvironments (figure 4B (see note added in proof (2)). The function of CD4+ DC within the B-cell areas and those surrounding the follicles, as well as the mechanism underlying the redistribution of these cells, is presently not known. It is possible that DC located in the follicles could directly interact with B cells and influence the initiation and regulation of antibody production [47]. The significance of the increase in both CD8α+ and CD8α–CD4– splenic DC in the red pulp following oral S. typhimurium infection (see note added in proof (2)) also remains to be determined. The accumulation of these cells early during infection, as well as their production of TNF-α, may precede their maturation and redistribution into the T-cell areas. This is consistent with studies showing that DC redistribute to T-cell areas in mice injected with LPS or an antigenic parasite extract [48–50], observations suggesting that DC respond spatially to antigenic challenge to become poised to prime T cells. The mechanism underlying the redistribution of DC into T-cell areas as well as the host and bacterial factors that modulate DC redistribution in response to Salmonella infection are presently unknown. Finally, the influence of DC subsets presenting Salmonella antigens on the subsequent T-cell response is also an important issue to be addressed. Elucidating the role of DC, and the identified DC subsets, in the adaptive immune response to Salmonella will provide valuable insight into the role of antigen-presenting cell populations in bacterial immunity.
Acknowledgments Work from our laboratory is funded by grants from the Swedish Natural Sciences Research Council, the European Commission, the Swedish Medical Research Council, the foundations of Österlund, Kock, Wiberg, Kungliga Fysiografiska and Crafoord, the Swedish Society for Medical Research, and Lund University Medical Faculty. The authors are grateful to past and present members of our research group, colleagues in our department as well as colleagues at other institutions that we are fortunate to have fruitful and exciting interactions with.
. Note added in proof 1. Yrlid U., Svensson M., Håkansson A., Chambers B.J., Ljunggren H.-G., Wick M.J., In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection, Infect. Immun. 69 (2001) 5726–5735. 2. Kirby A.C., Yrlid U., Svensson M., Wick M.J., Differential involvement of dendritic cell subsets during acute Salmonella infection, J. Immunol. 166 (2001) 6802–6811. 1246
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