Traditionally macrophages (MΦ) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MΦ process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MΦ and BMMΦ, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MΦ and BMMΦ use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMΦ by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMΦ. These data reveal that Salmonella co-incubation with BMDC as well as BMMΦ results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMΦ also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes naïve CD4+ and CD8+ T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.
The immune system has evolved to protect us from harmful microorganisms. To accomplish this complex task, two strategies are used, one providing non-specific protection and the other providing antigen-specific protection. The first line of defense, the innate immune system, consists of physical barriers such as skin, mucous membranes and secretions, that act in concert with phagocytic cells populating areas of antigen entry. The phagocytic cells non-specifically engulf invading microorganisms and eliminate them using an array of mechanisms including degradative enzymes as well as oxygen and nitrogen radicals ; the innate system does not have an enhanced response upon re-exposure to the same antigen. The second line of defense, the acquired immune system, has lymphocytes as a key component and generates a specific immune response. The hallmark features of the specific immune response are, as the name implies, antigen specificity as well as an ability to mount an enhanced response upon re-exposure to the same antigen. The innate immune system is linked to the specific immune system through a group of cells collectively called antigen presenting cells (APC). These cells function to take up foreign antigens such as bacteria, process the proteins into peptide fragments and present the peptides to T cells in the context of major histocompatibility complex (MHC) molecules. The presentation of antigens to T cells results in activation of effector lymphocytes and hence initiation of a specific immune response. Thus, phagocytosis and processing of invading pathogens by APC followed by presentation of pathogen-derived peptides to T cells is the key event in starting a specific immune response to microorganisms.
Antigen processing and presentation
The classical view of antigen processing and presentation is that two pathways exist. These pathways are defined largely by three characteristics: (1) the origin of the antigen being presented (endogenous vs. exogenous); (2) the type of MHC molecule loaded with peptides (MHC class I (MHC-I) vs. MHC class II (MHC-II) molecules); and (3) the subset of T cells stimulated (CD8+ (cytotoxic) T cells vs. CD4+ (helper) T cells) [2,3]. The pathways can be summarized as follows. Typically, MHC-I molecules are loaded with peptides derived from endogenous antigens synthesized within the APC. These antigens are processed within the cytosol by the proteasome, and the resulting peptides are transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). The peptides are loaded onto MHC-I molecules in the ER and the resulting peptide—MHC-I complexes are transported to the APC surface for recognition by CD8+ T cells . In contrast, exogenous antigens such as bacteria are internalized by APC and are processed into peptides in phagolysosomal compartments. Resulting peptides are loaded onto MHC-II molecules in compartments of the endosomal system and are transported to the cell surface for recognition by CD4+ T cells .
It has recently become evident, however, that these two pathways are not completely separated. Studies have shown that MHC-II molecules can present endogenous antigens to CD4+ T cells  and at least a subset of APC can process exogenous antigens for presentation by MHC-I molecules to CD8+ T cells [5–7]. This latter, alternative MHC-I antigen presentation pathway may be an important means by which the immune system detects and eliminates intracellular microorganisms residing in vacuolar compartments. It is this aspect of antigen processing and presentation, as well as T cell stimulation by microbes residing in vacuolar compartments, that is the focus of the present article. This article will discuss how bone marrow-derived macrophages (MΦ) (BMMΦ) and bone marrow-derived dendritic cells (DC) (BMDC) infected with the intracellular bacterium Salmonella typhimurium process the bacteria and present bacteria-derived antigens for T cell recognition. We will summarize the mechanisms by which BMMΦ and BMDC process S. typhimurium for peptide presentation on MHC-I and MHC-II, the influence of bacterial interactions with BMMΦ and BMDC on the expression of cell surface molecules important in triggering an immune response and on cytokine production by the infected APC. Finally, we will discuss the role of DC in priming an immune response to S. typhimurium infection using a murine model.
MΦ are bone marrow-derived cells that enter the blood as monocytes. After approximately a day in the blood stream, they enter and reside in several different tissues of the body including secondary lymphoid organs, lungs, skin and liver . MΦ are actively phagocytic and endocytic cells that express low levels of MHC-II and co-stimulatory molecules such as CD80 and CD86 unless activated by mediators such as interferon (IFN)-γ. The exposure of MΦ to inflammatory cytokines such as IFN-γ triggers a number of changes in the cells including upregulation of co-stimulatory molecules, increased endocytic capacity, secretion of pro-inflammatory cytokines and generation of reactive compounds that can destroy the phagocytosed pathogen [1,9]. Inflammatory stimuli also recruit large numbers of MΦ from the blood to the site of local inflammation. Due to their endocytic and phagocytic capacity as well as their ability to upregulate MHC and co-stimulatory molecules upon activation, MΦ have an important role as APC.
DC are also bone marrow—derived cells residing in organs exposed to antigen contact including the skin and mucous membranes. They are also important residents in other parts of the body such as lymphoid organs [10,11]. Unlike MΦ, which have a critical role in establishing inflammation, DC seem to have a more refined role as APC. This is due to several features of DC such as their ability to capture antigens, migrate to lymphoid organs and prime naïve T cells [10,11]. The function of DC, however, is linked to their differentiation state. Immature DC, i.e. cells that have not received antigenic stimulus, are highly active in internalizing antigens in soluble or particulate form. Immature DC use a variety of mechanisms to internalize antigens including pinocytosis, macropinocytosis, receptor-mediated internalization (FcγR or mannose receptor) or phagocytosis [12–15]. After receiving antigenic stimulus or being exposed to other ‘maturation’ signals such as IL-1β, TNF-α or LPS, the capacity of DC to internalize antigens is dramatically decreased and surface expression of co-stimulatory molecules including CD80 (B7-1), CD86 (B7-2), CD40, CD54 (ICAM-1) and both MHC-I and MHC-II is increased [13,14,16]. In addition, a rapid increase in biosynthesis and altered intracellular trafficking of MHC-II molecules is directly associated with DC maturation [17,18]. DC maturation also involves altered chemokine production by DC as well as modulation of chemokine receptor expression on DC that facilitates their migration from the periphery to the T cell areas of lymphoid organs [19,20]. Together, these observations have led to the following model for DC function as APC: immature DC acquire antigen in the periphery and begin to undergo a maturation program that downregulates antigen capture capacity and upregulates antigen presentation capacity. These processes, combined with modulation of chemokine production and chemokine receptor expression patterns, results in DC migration into lymphoid organs for interaction with antigen-specific T cells. Thus, DC expressing high levels of co-stimulatory molecules present peptides from antigens acquired in the periphery, thereby activating antigen-specific lymphocytes in the T cell areas of secondary lymphoid organs.
S. typhimurium is a Gram-negative bacterium that survives and replicates inside infected MΦ, remaining confined inside vacuoles in the infected cells . S. typhimurium has evolved a series of strategies to survive inside the harsh milieu of phagolysosomal compartments of phagocytic cells including delaying acidification of phagosomes containing bacteria, inducing tolerance to acid stress, and being resistant to a family of antimicrobial peptides called defensins . In addition, S. typhimurium is cytotoxic for the phagocytosing cell .
S. typhimurium infection causes typhoid fever-like pathogenesis in mice. The ingested bacteria cross the mucosal barrier by penetrating the M cells , invade the gut-associated lymphoid tissue and penetrate into spleen and liver . If untreated, the bacterial load in these organs increases due to replication inside phagocytic cells and death is the likely outcome of the infection. The pathogenesis of S. typhimurium in mice is similar to Salmonella typhi infection in humans, which has made this a useful animal model for studying typhoid fever pathogenesis.
Processing and presentation of bacterial antigens on MHC-I and MHC-II by infected APC
We have developed an in vitro system to study the ability of APC to process bacterial proteins by expressing well defined T cell epitopes as fusion proteins in Escherichia coli or S. typhimurium, bacteria that have no known mechanism of phagosomal escape. The initial studies showed that peritoneal-elicited MΦ could process and present bacterial antigens on MHC-II  as well as MHC-I . The observed presentation on MHC-II was predicted, since exogenous antigens such as bacteria are normally processed by APC in vacuoles of the endocytic system for peptide presentation on MHC-II. However, the MHC-I presentation of peptides derived from a model protein (Crl-OVA) expressed in the cytosol of bacteria that lack an ability to escape from phagosomal compartments was a novel finding . This MHC-I presentation required active uptake and processing of the bacteria, as viable MΦ were required for presentation of the MHC-I-binding OVA(257–264) peptide and inhibiting phagocytosis using cytochalasin D (CCD) abrogated the observed presentation. Furthermore, the MHC-I presentation was resistant to brefeldin A and cycloheximide, suggesting that a preformed pool of post-Golgi MHC-I was being used. In addition, it was shown that bacterial peptides were transferred from infected MΦ to preformed MHC-I molecules in a process termed peptide regurgitation . Further characterization of this alternative MHC-I presentation pathway revealed that the processing of the bacterial fusion protein by peritoneal-elicited MΦ was not dependent on TAP [30,31] or the proteasome . We have now extended these studies to BMMΦ which give identical results. That is, presentation of cytosolic OVA(257–264) by BMMΦ infected with E. coli expressing Crl-OVA does not depend on TAP (Fig. 1; ) or the proteasome . Thus, the activation status of the MΦ per se does not appear to alter the pathway used to present a model antigen expressed in E. coli with respect to dependence on TAP and the proteasome.
Considering the data demonstrating the phagocytic capacity of DC [12,33], we decided to investigate whether these cells were also able to process and present bacterial antigens on MHC-I and MHC-II. This was indeed demonstrated to be the case with BMDC cultured in GM-CSF for 6–8 days . These CD11c+ BMDC are in an intermediate stage of maturation . BMDC were shown to efficiently phagocytose E. coli expressing the model antigen Crl-OVA and process the bacteria for MHC-I and MHC-II presentation. Both MHC-I and MHC-II presentation required active phagocytosis of the bacteria and processing in vacuolar compartments. Bacteria processing by BMDC was rapid, requiring less than 30 min of DC bacteria co-incubation for significant peptide presentation on MHC-I . Similar rapid antigen processing for MHC-I presentation has also been measured for BMMΦ (Fig. 1). However, in contrast to the situation for bacteria-infected BMMΦ (Fig. 1), the pathway used by BMDC to present bacterial derived peptides on MHC-I was dependent on TAP as well as the proteasome, and uses newly synthesized MHC-I molecules . These initial studies were performed using E. coli expressing Crl-OVA, and we have also demonstrated that BMDC process S. typhimurium expressing Crl-OVA for presentation of the OVA(257–264) epitope on Kb using a TAP-dependent pathway (Fig. 2).
Thus, both BMMΦ and BMDC are efficient at processing and presenting bacterial antigens on MHC molecules but the pathways used to accomplish this are not identical:
BMMΦ and peritoneal-elicited MΦ can process and present bacterial antigens on MHC-II but the cells need to be stimulated with IFN-γ to induce sufficient levels of MHC-II expression. BMDC, however, constitutively express high levels of MHC-II and therefore do not require IFN-γ pretreatment to present antigens on MHC-II.
BMDC require newly synthesized MHC-I, the proteasome and the TAP transporter to present bacterial antigens on MHC-I, showing that the cytosolic pathway is preferentially used. In contrast, BMMΦ and peritoneal-elicited MΦ use a non-cytosolic pathway as well as the cytosolic pathway for presenting bacterial antigens on MHC-I. BMMΦ can generate MHC-I loaded with bacteria-derived peptides without using newly synthesized MHC-I molecules, and do not require the proteasome or TAP. It should be noted that although BMMΦ presentation on MHC-I occurs in the absence of a functioning cytosolic MHC-I presentation pathway, it is reduced compared to the level of presentation when the cytosolic pathway is intact, at least as measured in experiments of 90 min or longer duration [30–32]. Shorter incubations (30 min or less), such as that shown in Fig. 1, reveal equal levels of presentation. This initial, rapid presentation may be due to the peptide regurgitation pathway, while at later time points, the cytosolic pathway may contribute significantly. This suggests that both cytosolic as well as non-cytosolic pathways are utilized in BMMΦ infected with E. coli or S. typhimurium and that initially the non-cytosolic is the predominant pathway detected.
BMDC do not regurgitate peptides that bind preformed MHC-I molecules following phagocytosis of E. coli. In contrast, BMMΦ and peritoneal-elicited MΦ regurgitate peptides that bind MHC-I molecules following phagocytosis of E. coli (data not shown and ). The explanation for this difference in peptide regurgitation is not clear, and could be due to different characteristics of the cells. For example, BMDC phagocytose E. coli at a slower rate than BMMΦ, and BMDC accumulate fewer bacteria per cell compared to BMMΦ. Other contributing factors could be different degradative capacities of the endosomal/phagolysosomal compartments harboring the bacteria in BMDC vs. BMMΦ or differences in trafficking of bacteria-containing vacuoles in the two types of APC.
Cell surface molecule expression and cytokine production by bacteria-infected APC
Immunomodulatory compounds such as TNF-α or LPS induce maturation in DC [13,14,16] and cause changes in the properties of MΦ. LPS is a component of the cell surface of Gram-negative bacteria and accordingly, the influence of Gram-negative bacteria on DC maturation as well as on MΦ is an interesting issue to address. We have shown that co-incubation of BMDC or BMMΦ with S. typhimurium results in upregulation of a number of cell surface antigens including CD80, CD86, MHC-I and MHC-II (Fig. 3; Svensson et al., in preparation). This response does not require internalization of the bacteria, nor does it require that the bacteria are viable, since similar upregulation of the surface molecules is observed upon co-culture of APC and bacteria in the presence of CCD or with heat-killed bacteria. An influence of S. typhimurium and E. coli on DC is consistent with other reports demonstrating reduced endocytic capacity after phagocytosis of Chlamydia psittaci and enhanced synthesis and stability of MHC-I and MHC-II molecules after infection with the Gram-positive bacteria Streptococcus gordonii.
We have also shown that co-incubation of APC with S. typhimurium or E. coli results in IL-6 and IL-12 production by APC (Table 1). However, unlike the conditions that resulted in upregulation of surface molecules as discussed above, significant cytokine production by BMDC and BMMΦ requires phagocytosis of the bacteria, as CCD treatment of APC results in only moderate levels of cytokine secretion. In addition, although bacterial viability is not absolutely required for cytokine production by infected BMDC, the quantity of cytokine detected is significantly enhanced by co-incubation of APC with viable bacteria (Table 1). This is in contrast to the case for bacteria-infected BMMΦ, which produce approximately the same amount of IL-6 or IL-12 using heat-killed or viable bacteria (Table 1). Finally, co-incubation of BMMΦ with E. coli or S. typhimurium induces nitric oxide synthase (iNOS) and production of reactive nitric oxide intermediates, as assessed by quantitating the accumulation of NO2− (Table 1). BMDC infected with bacteria, however, do not stimulate iNOS and do not produce significant amounts of reactive nitric oxide intermediates (Table 1). This is yet another feature that distinguishes the different response of BMDC and BMMΦ to infection with Gram-negative bacteria.
|S. typhimurium (heat-killed)||−||+||+|
|1 µm Polystyrene beads||−||−||−|
|S. typhimurium (heat-killed)||−||++||++|
|1 µm Polystyrene beads||−||−||−|
|S. typhimurium (heat-killed)||−||+||+|
|1 µm Polystyrene beads||−||−||−|
|S. typhimurium (heat-killed)||−||++||++|
|1 µm Polystyrene beads||−||−||−|
Bone marrow from C56BL/6 mice was cultured for 7 days in IMDM media supplemented with GM-CSF. DC or MΦ derived from the bone marrow cultures were co-incubated with either viable or heat-killed (65°C, 40 min) S. typhimurium 14028 (15:1 ratio) in either the absence or presence (+CCD) of CCD (10 µg ml−1; to inhibit phagocytosis) for 2 h. The remaining bacteria were subsequently washed away, cells were treated with 25 µg ml−1 gentamicin, and were cultured for an additional 46 h. At this time point, the presence of cytokines in the supernatants was determined by an enzyme-linked immunosorbent assay using antibody pairs specific for either IL-12 or IL-6. The presence of nitrite in the supernatants was also determined by mixing 100 µl of supernatant 1:1 with Griess reagent (1% sulfanilamide, 1% naphthylethyne diamine dihydrochloride, 2.5% H3PO4), incubating at room temperature for 10 min and reading the absorbance at 540 nm. NaNO2 was used as the standard for nitrite determinations.
Assessing the role of DC to Salmonella infection in vivo
The studies discussed above have shown that BMDC phagocytose bacteria and process them for peptide presentation on MHC-I and MHC-II. In addition, BMDC interaction with bacteria results in altered expression of co-stimulatory molecules important in triggering the immune response and in cytokine production by the infected cells. However, it is not yet clear what role DC play in initiating or maintaining an immune response to bacterial infection in vivo. Do bacteria such as S. typhimurium reside in DC in vivo? If so, what is the role of these infected DC in influencing the immune response to the bacteria? To initiate studies to address these issues, we have loaded BMDC with S. typhimurium expressing a model antigen in vitro under conditions where the BMDC present bacterial derived peptides on MHC-I and MHC-II. Injection of Salmonella-loaded DC into mice results in activation of both CD4+and CD8+ T cells that are specific for the Salmonella-encoded model antigen. This was assessed by demonstrating upregulation of CD44 expression on both CD4+ and CD8+ T cells as well as the presence of antigen-specific cytotoxic T cells and IFN-γ-producing CD8+ T cells in the spleens and mesenteric lymph nodes of DC-immunized mice (Yrlid et al., submitted). In the splenic APC population, host APC, primarily DC, are activated. This was shown by the presence of a population of CD11c+ cells expressing higher amounts of the co-stimulatory molecules CD40, CD80 and CD86 (Yrlid et al., submitted). These results suggest that DC-containing S. typhimurium can stimulate naïve T cells. This is supported by another study showing that DC loaded in vitro with non-viable Chlamydia trachomatis presented antigen to Chlamydia-sensitized CD4+ T cells . The Chlamydia-loaded DC also induced protective immunity against chlamydial infection in the genital tract that was equal to that obtained after infection with live bacteria. Together, these results suggest that DC have a role in initiating an immune response to infection with Gram-negative bacteria.
In the immune response to a bacterial infection, MΦ play a critical role by phagocytosing and destroying the microorganisms as well as presenting bacteria derived peptides to T cells. These APC also produce cytokines such as IL-6 and IL-12 which can influence the subsequent immune response. It has recently become evident that DC are also phagocytic and can process bacterial antigens for peptide presentation on MHC-I and MHC-II. Like MΦ, DC also produce cytokines in response to bacterial infection, bacterial products such as LPS or other immunomodulatory cytokines including TNF-α. Data are also beginning to emerge showing that DC have a role in the immune response to bacteria  and parasite [38,39] infections in vivo. However, the precise contribution of DC vs. MΦ to microbial infections awaits appropriate animal models, such as knock out mice specifically lacking one or the other type of APC or a means to specifically ablate MΦ or DC while leaving the other population unperturbed. In this article, we have discussed data generated in our laboratory, both in vitro and in vivo, as well as data published by several groups demonstrating that in addition to MΦ, DC also play a pivotal role in the immune response to microbial infections.
There are important functional similarities between these APC. Both MΦ and DC are present in the periphery and are highly phagocytic (however, DC only in their immature state). Both APC types respond to Gram-negative bacteria by secreting IL-6 and IL-12. The production of IL-12 by phagocytic cells may be of particular importance in mounting cell-mediated responses against pathogens such as S. typhimurium that reside and replicate in vacuolar compartments of phagocytic cells. Similar to BMMΦ and peritoneal-elicited MΦ, DC are capable of processing bacterial antigens for peptide presentation on MHC-I and MHC-II. Furthermore, bacterial interaction with either BMMΦ or BMDC results in upregulation of several co-stimulatory molecules important for T cell activation. Immunization of mice with DC that have phagocytosed S. typhimurium generates cytotoxic T cells and IFN-γ-producing CD8+ T cells specific for bacteria-encoded antigens. This demonstrates that BMDC can prime naïve T cells to bacterial antigens in vivo.
Functional differences between MΦ and DC may explain more about their specific role in generating an immune response to microbes. For example, BMDC are poor producers of nitric oxide radicals after bacterial infection whereas MΦ are robust inducers of iNOS and produce reactive nitrogen intermediates (Table 1; Svensson et al., in preparation). The functional significance of this differential iNOS induction between MΦ and DC remains to be studied, but it could have consequences for microbial survival, antigen processing and presentation, or bacterial persistence in vivo. Moreover, the different level of co-stimulatory molecules expressed by DC vs. MΦ may influence the ability of the APC types to stimulate a T cell response. Whereas DC have moderate to high levels of MHC-I, MHC-II, CD40 and CD86 constitutively expressed on their surface, MΦ constitutively express a high level of only MHC-I; MΦ must be activated to increase expression of MHC-II and CD86, for example. The high migratory capacity of DC relative to MΦ is also a significant difference between the two types of APC; DC have a coordinated program of chemokine production and chemokine receptor modulation that corresponds with maturation after antigen exposure or stimulation by immunomodulatory compounds. The features of DC and MΦ described here may provide insight into their relative roles in the immune response to bacterial pathogens. However, defining the relative role of these two types of APC to microbial infection in vivo awaits further definitive studies.
This work was supported by the Swedish Natural Sciences Research Council (project 10610-306), the Swedish Foundation for Strategic Research Infection and Vaccinology Program, Lund University Medical Faculty, The Österlund Foundation, Kock's Foundation, Kungliga Foundation, The Crafoord Foundation, and Åke Wiberg's Foundation.