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. 2007 Mar;120(3):424–432. doi: 10.1111/j.1365-2567.2006.02527.x

Fcγ receptors are crucial for the expression of acquired resistance to virulent Salmonella enterica serovar Typhimurium in vivo but are not required for the induction of humoral or T-cell-mediated immunity

Nathalie Menager 1, Gemma Foster 1, Sanja Ugrinovic 1,3, Hazel Uppington 1, Sjef Verbeek 2, Pietro Mastroeni 1
PMCID: PMC2265895  PMID: 17328787

Abstract

Antibodies play an important role in immunity to Salmonella enterica. Here we evaluated the requirement for Fcγ receptors in host resistance to S. enterica using an in vivo model of systemic infection. We show that mice lacking FcγRI, II and III can control and clear a primary infection with S. enterica micro-organisms of low virulence, but are impaired in the expression of vaccine-induced acquired immunity to oral challenge with virulent bacteria. We also show that, in vivo, FcγRI, II, III−/− mice were able to mount efficient T-helper 1 type T-cell responses and antibody responses specific for S. enterica. The work indicates that targeting S. enterica to FcγR is needed for the expression of vaccine-induced acquired immunity, but is not essential for the engenderment of T- and B-cell immunity to the bacterium in vivo.

Keywords: antibody, Fc receptors, immunity, infection, Salmonella

Introduction

Salmonella enterica is generally believed to be an intracellular pathogen1,2 that grows preferentially within phagocytes and can evade killing by these cells.3 During systemic infections, bacterial growth in the infected tissues is controlled by resident and inflammatory phagocytes that are recruited to the foci of infection and are activated via the production of inflammatory cytokines [tumour necrosis factor-α (TNF-α), interleukin-12, (IL-12), IL-18, interferon-γ (IFN-γ), IL-15].4–13 T cells and antibody do not appear to be essential for the control of bacterial growth in the early stages of S. enterica systemic infections.14,15 However, T cells contribute to the clearance of the bacteria from the tissues in the late stages of the primary disease.16,17 The concerted action of both anti-S. enterica antibody and T cells is needed for the expression of a high level of resistance to secondary infections with virulent pathogens in vaccinated individuals.18,19

The requirement for antibody in the expression of host resistance to S. enterica implies that the bacteria are, at least transiently, present in the extracellular compartment. In fact, bacteraemia is a common feature of systemic S. enterica infections of both animals and humans.20–22 Furthermore, during their growth S. enterica spread from infected phagocytes to uninfected ones, presumably via the extracellular space.1,23 Opsonization by specific antibodies in the extracellular compartment may facilitate the uptake of the bacteria by phagocytes and possibly up-regulate their antimicrobial functions. This could be mediated by binding of antibody-opsonized bacteria directly to Fc receptors (FcR) or to complement receptors. Mice express three receptors for immunoglobulin G (IgG), FcγRI, FcγRII and FcγRIII. Two of these are activating receptors (FcγRI and FcγRIII) that signal via two membrane-bound γ-chains containing immuno-receptor tyrosine-based activation motifs (ITAM) and one is an inhibitory receptor (FcγRII) that signals through an immuno-receptor tyrosine-based inhibitory motif (ITIM) resulting in the inhibition of many of the functions activated by FcγRI and FcγRIII.24 A fourth FcγR receptor has also been reported.25,26

Macrophages can either kill or restrain the replication of intracellular S. enterica by lysosomal enzymes, production of reactive oxygen intermediates, reactive nitrogen intermediates and antimicrobial peptides.27,28 We have recently reported that opsonization of S. enterica with serum collected from vaccinated animals enhances the uptake of the bacteria by phagocytic cells in vitro via stimulation of FcγRI. This results in increased production of reactive oxygen intermediates leading to an increase in the antibacterial functions of the infected cells.29 Despite the in vitro evidence showing that opsonization with antibody enhances bacterial killing by phagocytes, the role of FcγR in immunity to S. enterica in vivo is still unclear. It is still unknown whether FcγR are essential for host resistance to S. enterica in vivo or whether its function in vivo is rendered redundant by the presence of other receptors (e.g. complement receptors). This has been investigated in the present paper.

Materials and methods

Reagents and media

All reagents and media were obtained from Sigma-Aldrich, Poole, UK unless stated otherwise.

Mice

Slc11a1–/– FcγRI–/– FcγR2–/– FcγR3–/– mice (FcγRI–/– FcγRII–/–FcγRIII–/–) lacking simultaneously FcγRI, FcγRII and FcγRIII and wild-type control mice on a 129Ola/C57BL/6 background were used. Controls matched for strain, age and sex were used in all experiments. The mice were bred in the Cambridge animal unit from breeding pairs generated by Dr J. S. Verbeek, University of Leiden, the Netherlands.

Bacterial strains

Salmonella enterica serovar Typhimurium SL3261 is an attenuated aroA derivative of the wild-type SL1344 strain with an intravenous (i.v.) 50% lethal dose (LD50) for Slc11a1–/– mice of approximately 107 colony-forming units (CFU).30Salmonella enterica serovar Typhimurium C5 is a virulent strain with an i.v. LD50 of < 10 CFU for Slc11a1–/– mice.31 For intravenous inoculation, bacteria were grown at 37° as stationary overnight cultures in Luria–Bertani broth and diluted in phosphate-buffered saline (PBS). For oral challenge, the mice were lightly anaesthetized and the bacteria were administered intragastrically via a gavage tube. The inoculum was checked by plating on Luria–Bertani agar.

Enumeration of viable S. enterica in the tissues

Mice were killed by cervical dislocation. Their spleens and livers were homogenized in a Seward Stomacher 80 Biomaster (Seward, Worthing, UK) in 10 ml distilled water. Viable bacterial counts were assayed on pour plates of Luria–Bertani agar.28 Viable bacterial counts are presented as log10 mean ± SD of CFU.

Preparation of T-cell antigen from S. enterica

Soluble extract antigen (C5/NaOH) was prepared from cultures of S. enterica serovar Typhimurium strain C5 as described previously.32 Briefly, an overnight stationary culture of strain C5 in Luria–Bertani broth was pelleted, washed once in PBS containing 5 mm ethylenediaminetetraacetic acid, and washed once more in PBS. The bacteria were sonicated on ice. Cellular debris was removed by centrifugation at 13 000 g for 20 min. The supernatant was filtered through a 0·22-μm pore-size filter (Sartorius, Epsom, UK) and stored at − 70°. Alkali-treated antigen (C5/NaOH) was prepared by the addition of NaOH up to 0·25 m; the mixture was incubated at 37° for 3 hr before it was neutralized with HCl and filtered. The protein concentrations of the antigens were determined by using a bicinchoninic acid kit (Pierce Biochemicals, Rockford, IL) according to the manufacturer's instructions.

Antibodies, tissue culture reagents and cell lines

Mouse monoclonal antibodies to CD16/CD32 (purified), T-cell receptor-β (TCR-β), CD3, CD4, CD8, CD19, CD11b, CD11c, CD69 and IFN-γ, isotype controls, and other reagents used for flow cytometry and intracellular cytokine staining were purchased from BD PharMingen (Cowley, UK). Unless otherwise stated, antibodies were directly conjugated to fluorescein isothiocyanate, phycoerthythrin, or Cy-Chrome.

The following reagents were used for tissue culturing: phorbol 12-myristate 13-acetate (PMA) (5 ng/ml), ionomycin (1·25 μm; Sigma), mitomycin C (25 μg/ml), and anti-CD28 (1 μg/ml). All cells were cultured in RF10 complete medium, consisting of RPMI-1640 supplemented with 10% fetal bovine serum, 2 mm glutamine and 0·05 mm 2-mercaptoethanol.

CD4+ T cells were positively enriched with magnetic bead-conjugated antibodies as instructed by the manufacturer (Miltenyi Biotec, Camberley, UK). CD4+ T cells were found to be > 95% pure as assessed by flow cytometry with a FACSCalibur flow cytometer (Becton Dickinson, Oxford, UK).

Detection of anti-lipopolysaccharide antibodies

Lipopolysaccharide (LPS) from S. enterica serovar Typhimurium (Sigma) was used at a concentration of 5 μg/ml to coat the wells of immunoplates.33 Twofold dilutions of mouse sera in PBS, 0·05% Tween 20, 1% bovine serum albumin were added in 50 μl to the wells in triplicate. The plates were washed after a 2-hr incubation. Total antibody was detected with horseradish peroxidase-conjugated goat anti-mouse antibody (Dako, High Wycombe, UK). To detect antibody isotypes and subclasses, horseradish peroxidase-conjugated anti-mouse IgM, IgG or IgG subclass antibodies (Sigma) were used. The plates were developed with O-phenylenediamine (OPD) substrate (Sigma) and absorbance was read at 490 nm.

Cytokine measurement by enzyme-linked immunosorbent assay (ELISA)

Positively enriched splenic CD4+ T cells were stimulated with 10 μg/ml C5/NaOH in the presence of mitomycin C-treated (30 min at 37°) splenic antigen-presenting cells. Levels of IFN-γ and IL-2 produced by CD4+ T cells were detected using ELISA Duoset kits following the manufacturer's instructions (R & D Systems Europe Ltd, Abingdon, UK).

Intracellular cytokine staining

Intracellular cytokine staining for IFN-γ was performed with the Cytofix/Cytoperm Plus kit as instructed by the manufacturer (BD PharMingen). Briefly, splenocytes (5 × 106 cells/ml) were stimulated with either medium alone, C5/NaOH (10 μg/ml), or PMA and ionomycin in the presence of 1 μg anti-CD28 antibody/ml. Brefeldin A (1 μl/ml) was added 6 hr after the initiation of the culture. Cells were collected 18 hr later and stained for surface markers (CD4, CD8 and CD69). Cells were fixed for 30 min, washed, permeabilized for 10 min, washed again, and incubated with anti-IFN-γ antibodies for 30 min. Cells were washed and analysed with a FACSCalibur flow cytometer. Cytokine-positive CD4+ or CD8+ cells that were also positive for the activation marker CD69 were counted. The percentages of cytokine-positive cells detected when cultured in the presence of antigens, corrected for background levels measured in the absence of antigens, are presented here.

Statistical analysis

Student's t-test was used to determine the significance of differences between controls and experimental groups. Differences between experimental groups were considered significant for P-values < 0·05.

Results

Bacterial numbers in the spleen and liver after immunization with S. enterica serovar Typhimurium SL3261

FcγRI–/– FcγRII–/– FcγRIII–/– and wild-type control 129Ola/C57BL/6 mice were immunized i.v. with 5 × 105 CFU of the live attenuated S. enterica serovar Typhimurium SL3261 strain. Viable bacterial counts were performed on spleen and liver homogenates from groups of five mice per time point thereafter. Bacterial counts were significantly higher (P < 0·05) in the spleens of FcγRI–/– FcγRII–/– FcγRIII–/– mice on days 3 and 7 after infection compared with control animals (Fig. 1a). Liver counts were higher in the mutant animals on days 1, 3 and 7 after infection (Fig. 1b). In both groups of mice, bacterial numbers started to decline after day 15 and low bacterial counts were seen by day 56. Complete clearance of the infection was achieved by week 10 after immunization (not shown). A repeat experiment gave similar results (not shown). The FcγRI–/– FcγRII–/– FcγRIII–/– mice had slightly higher bacterial counts than wild-type mice early in the infection but could efficiently eliminate the bacteria from the spleen and liver thereafter.

Figure 1.

Figure 1

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FcγRI–/– FcγRII–/– FcγRIII–/– mice (•) and wild-type control 129Ola/C57BL/6 mice (□) were infected with 5 × 105 CFU S. enterica serovar Typhimurium strain SL3261. Spleen (a) and liver (b) counts of viable bacteria were obtained thereafter. Results are expressed as mean ± SD from groups of five mice. *Denotes P < 0·05 when comparing FcγRI–/–, FcγRII–/–, FcγRIII–/– mice with wild-type control 129Ola/C57BL/6 mice.

FcγRI–/– FcγRII–/– FcγRIII–/– mice immunized with S. enterica serovar Typhimurium SL3261 fail to control an infection with virulent bacteria

Groups of 10 FcγRI–/– FcγRII–/– FcγRIII–/– mice and wild-type control 129Ola/C57BL/6 mice were immunized with S. enterica serovar Typhimurium SL3261 as in Fig. 1. A similar number of age-matched and sex-matched naive mice for each strain were kept alongside as unimmunized controls. Ten weeks after vaccination, all mice were challenged orally with approximately 5 × 109S. enterica serovar Typhimurium C5. As expected, all the naive mice of either strain succumbed to the virulent challenge within 7 days after infection. Only 10% of the immunized 129Ola/C57BL/6 mice succumbed to the infection; conversely, 80% of immunized FcγRI–/– FcγRII–/– FcγRIII–/– mice died within 35 days of challenge (Fig. 2). A repeat experiment gave very similar results (not shown). Thus, FcγRI–/– FcγRII–/– FcγRIII–/– mice fail to express long-term vaccine-induced immunity to S. enterica.

Figure 2.

Figure 2

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FcγRI–/– FcγRII–/– FcγRIII–/–mice (•) and wild-type control 129Ola/C57BL/6 mice (□) were infected with 5 × 105 CFU S. enterica serovar Typhimurium strain SL3261. Age-matched naive FcγRI–/– FcγRII–/– FcγRIII–/– mice (○) and naive wild-type control 129Ola/C57BL/6 mice (□) were used as unimmunized controls. Ten weeks after vaccination, all mice were challenged orally with 5 × 109 CFU of virulent serovar Typhimurium strain C5. Results are expressed as percentage of survivors from groups of 10 mice.

Immune responses in FcγRI–/– FcγRII–/– FcγRIII–/– mice immunized with S. enterica serovar Typhimurium SL3261

The lack of vaccine-induced protection observed in the FcγRI–/– FcγRII–/– FcγRIII–/– mice could be the result of the impairment of FcγR-mediated effector functions of phagocytic cells or alternatively to the failure of these mutant mice to mount protective anti-S. enterica-acquired antibody and/or T-cell-dependent immunity. In fact, binding of antigen–antibody complexes to FcγRs can result in more efficient antigen presentation by dendritic cells and thus increased immunogenicity.34–36In vitro studies have indicated that the targeting of opsonized S. enterica to FcγR can enhance antigen presentation of S. enterica antigens by dendritic cells to T cells because the opsonized bacteria avoid lysosomal degradation.37 To address whether the known FcγR-mediated enhancement of antigen presentation to T cells affects the in vivo activation or persistence of acquired immunity to S. enterica, we have monitored key parameters of T- and B-cell responses that are required for resistance to salmonellosis.38,39 FcγRI–/– FcγRII–/– FcγRIII–/– mice and wild-type control 129Ola/C57BL/6 were vaccinated i.v. as in Fig. 1. Anti-S. enterica serum antibodies and T-cell responses were tested at 3, 6 and 9 weeks post immunization.

Anti-S. enterica antibodies

Total serum anti-LPS immunoglobulins were measured by ELISA in the sera of immunized mice at weeks 3, 6 and 9 after immunization. FcγRI–/– FcγRII–/– FcγRIII–/– mice had higher levels of total anti-S. enterica LPS immunoglobulin compared to 129Ola/C57BL/6 mice at weeks 3 and 6 after immunization, with similar levels being found in the two mouse strains at 9 weeks (Fig. 3a). The presence of anti-LPS IgG responses in mice vaccinated with live attenuated S. enterica vaccines is associated with the concomitant presence of T-cell immunity to the bacterium.38 We therefore measured IgG subclasses in immunized mutant and control animals. Compared to 129Ola/C57BL/6 mice, FcγRI–/– FcγRII–/– FcγRIII–/– mice had higher levels of IgG2a at 3, 6 and 9 weeks after immunization, but only higher levels of total IgG1 and IgG2b at 3 weeks (Fig. 3b). IgM and IgG3 levels were similar in both strains of mice at all the time-points tested (not shown). Thus, FcγRI–/–FcγRII–/– FcγRIII–/– mice do not appear to be impaired in their ability to develop antibody responses to S. enterica and have higher level of circulating anti-S. enterica LPS IgG in the early stages of a primary infection.

Figure 3.

Figure 3

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FcγRI–/– FcγRII–/– FcγRIII–/– mice (•) and wild-type control 129Ola/C57BL/6 mice (□) were immunized with 5 × 105 CFU of serovar Typhimurium strain SL3261 as for Fig. 1 (a). Total serum anti-LPS antibodies were measured by ELISA in the sera of mice 3, 6 and 9 weeks after immunization. Results are expressed as optical density (OD) readings (at 490 nm) from groups of seven mice. The bar represents the mean for each mouse strain. (b) Anti-LPS IgG1, IgG2a and IgG2b subclasses were measured by ELISA in the sera of mice 3, 6 and 9 weeks after immunization. Results are expressed as OD readings (at 490 nm) from groups of seven mice. *Denotes P < 0·05 when comparing FcγRI–/– FcγRII–/– FcγRIII–/– mice with wild-type control 129Ola/C57BL/6 mice.

T-cell responses

To evaluate whether there was impairment in the induction and persistence of T-cell responses in mice lacking FcγRI, FcγRII and FcγRIII, we immunized groups of mutant and control animals as above and monitored T-cell responses in the spleen. Figure 4 shows that, at 3, 6 and 9 weeks after immunization, the percentage of CD4+ CD69+ and CD8+ CD69+ cells capable of producing IFN-γ in response to stimulation with the S. enterica antigen extract C5/NaOH were similar in FcγRI–/–FcγRII–/– FcγRIII–/– mice and wild-type control 129Ola/C57BL/6 mice. Purified CD4+ T cells from FcγRI–/–FcγRII–/– FcγRIII–/– mice and 129Ola/C57BL/6 mice were able to release similar amounts of IL-2 in response to C5/NaOH at 3 and 6 weeks after immunization; a small reduction in IL-2 production was observed from CD4+ T cells of FcγRI–/– FcγRII–/– FcγRIII–/– mice only at 9 weeks after vaccine administration (Fig. 5a). IFN-γ was produced by CD4+ T cells from FcγRI–/– FcγRII–/– FcγRIII–/– mice and 129Ola/C57BL/6 mice at similar levels 6 and 9 weeks after immunization with a transient difference in IFN-γ levels at 3 weeks (Fig. 5b). Thus, administration of live attenuated S. enterica induces long-lasting T helper type 1 T-cell responses in both FcγRI–/– FcγRII–/–FcγRIII–/– mice and 129Ola/C57BL/6 mice.

Figure 4.

Figure 4

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FcγRI–/– FcγRII–/– FcγRIII–/– mice (closed bars) and wild-type control 129Ola/C57BL/6 mice (open bars) were immunized with 5 × 105 CFU of serovar Typhimurium strain SL3261 as for Fig. 1. Whole splenocytes were obtained from mice killed at 3, 6 and 9 weeks post-immunization and stimulated with C5/NaOH in the presence of anti-CD28 antibodies and brefeldin A and stained for IFN-γ expression as indicated in the materials and methods. CD4+ (a) and CD8+ (b) T cells were gated, and the percentages of IFN-γ-positive T cells that were also CD69+ were calculated. Results are expressed as mean ± standard deviation of the cell percentages from groups of three mice. The percentages of cytokine-positive cells in naive mice were < 0·6% and < 0·4% for CD4+ and CD8+ cells, respectively.

Figure 5.

Figure 5

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FcγRI–/– FcγRII–/– FcγRIII–/– mice (closed bars) and wild-type control 129Ola/C57BL/6 mice (open bars) were immunized with 5 × 105 CFU of serovar Typhimurium strain SL3261 as for Fig. 1. Levels of IL-2 (a) and IFN-γ (b) were measured by ELISA in the supernatants of CD4+ T cells purified from the spleens of mice 3, 6 and 9 weeks after immunization and stimulated with with C5/NaOH (10 μg/ml). Results are expressed as mean ± standard deviation for groups of three mice. *Denotes P < 0·05 when comparing FcγRI–/– FcγRII–/– FcγRIII–/– mice with wild-type control 129Ola/C57BL/6 mice.

Discussion

In the present paper we show that mice lacking FcγRI, FcγRII and FcγRIII can control and clear a primary infection by S. enterica micro-organisms of low virulence, but are impaired in their expression of vaccine-induced acquired immunity to oral challenge with virulent bacteria. This is likely to be the result of the lack of effector functions normally mediated by FcγR. In fact, the mutant mice can mount T-cell and B-cell (antibody) responses to S. enterica.

In the early stages of primary infections, the growth of S. enterica is controlled by resident and inflammatory phagocytes organized within discrete foci2,40–42 with the contribution of cytokines including TNF-α, IL-12, IL-18, IFN-γ and IL-15.4–13 The suppression of the early growth of S. enterica in the spleen and liver in primary infections does not require functional T and B cells (or antibodies) being largely mediated by an influx of cytokine-activated mononuclear cells.14,15 In the present study, we reported that the absence of FcγRI, FcγRII and FcγRIII does not abrogate the ability of mice to bring under control the primary infection. This is consistent with the notion that antibodies are dispensable for host survival in primary infections. However, the small differences in the bacterial burden that could be reproducibly seen in FcγRI–/–FcγRII–/– FcγRIII–/– and control mice indicate that antibody-binding to cell surface FcγR makes an appreciable contribution to the mechanisms responsible for the restraint of the growth of S. enterica in the spleen and liver during primary infections. These differences are seen very early after infection, when levels of anti-Salmonella IgG would be negligible, and so it is possible that the differences in bacterial burden between wild-type and mutant mice are the result of pre-existing (e.g. natural) antibodies capable of binding to the surface of the bacteria.

The role of antibody in resistance to salmonellosis becomes very apparent in secondary infections with virulent bacteria. In this scenario, T cells alone can confer only a low level of resistance, both anti-S. enterica antibodies and T cells (CD4+ and CD8+) being needed for host survival.18 The requirement for FcγRI, FcγRII and FcγRIII in the expression of host resistance in secondary infections indicates that FcγR are indispensable mediators of the protective effects of anti-S. enterica immunoglobulin. The results indicate that the function of FcγR is not redundant and their absence in secondary infections cannot be entirely compensated by the presence of other opsonic receptors on phagocytes, such as complement receptors. We have also found that C3-deficient or C1q-deficient mice immunized with live attenuated S. enterica are protected against oral challenge with wild-type bacteria (data not shown). This indicates that, unlike FcγR, the C3-dependent and C1q-dependent complement pathways are dispensable for acquired resistance to S. enterica.

The requirement for antibody binding to FcγR as a key effector mechanism operating in resistance to salmonellosis indicates that S. enterica during its growth is located transiently in the extracellular space. Interestingly, bacteraemia is a common feature of systemic S. enterica infections and antibiotics (i.e. gentamicin) that poorly penetrate phagocytes can affect the numbers of S. enterica in the tissues of infected mice.43 More to the point, we have recently found that the growth of S. enterica in the tissues is closely paralleled by increases in the number of infected phagocytes. This is likely to result from the release of the bacteria from heavily infected phagocytes into the extracellular space, leading to infection of new phagocytes.1,23 In the extracellular space, the bacteria would be targeted by opsonizing antibody and would interact with other phagocytes binding to surface FcγR leading to the enhancement of antibacterial phagocyte functions. The in vivo results presented in this paper parallel and extend our recently published data that indicated an important role for FcγR (mainly FcγRI) in the enhancement of S. enterica internalization by cultured macrophages and in the enhancement of antibacterial functions of the infected cells via the production of reactive oxygen intermediates.29 A role for FcγR has also been shown in vivo in host resistance to Bordetella pertussis infections, where antibodies have been documented to play an important role in protection against disease.44 Our present and past work has highlighted the requirement of antibodies and FcγR (in addition to T-cell-mediated immunity) in host resistance to S. enterica12,18,19,29 and has shown that bacteria continuously spread from uninfected to infected cells during their growth in the tissues.1 This work has provided a more comprehensive view of the pathogenesis of these infections and has provided useful information on the immunological requirements for the prevention of S. enterica infection. It is now clear that optimal control of S. enterica infections (e.g. via improved vaccination strategies) not only needs to restrain bacterial growth within individual phagocytic cells but also requires the generation of antibodies capable of targeting high-affinity FcR, such as FcγRI.

In addition to activating immune effector functions of phagocytic cells, the interaction of antigens with FcγR and the complement system can modulate the engenderment and/or development of specific immunity. This can be achieved through a number of mechanisms including, for example, enhancement of antigen presentation, lowering the activation threshold of immune cells and prolonging the persistence of antigen on follicular dendridic cells in the germinal centres.45–49 We have reported that B-cell-deficient mice are unable to mount protective T helper type 1 T-cell responses to S. enterica. Recently, targeting S. enterica to FcγR on dendritic cells in vitro has been shown to overcome the ability of the bacteria to avoid antigen presentation by these cells and to augment antigen processing and presentation to T cells.37 Taken together these findings may suggest the possibility that antibodies may make a major contribution to the engenderment of T-cell responses to the bacterium during vaccination via binding to FcγR and enhancing immune responses.50,51 However, in the present report we show that, in vivo, FcγRI–/– FcγRII–/– FcγRIII–/– mice were able to mount efficient T helper type 1 and IgG responses specific for S. enterica. This indicates that targeting S. enterica to FcγRI–/– FcγRII–/– FcγRIII–/– is not required for the engenderment of T-cell and B-cell immunity to the bacterium in vivo. It is thus possible either that targeting bacteria to antigen-presenting cells via antibody in vivo does not significantly modulate the development of acquired immunity to S. enterica, or that other cell surface receptors (e.g. complement receptors) can also enable antibody-coated bacteria to augment T-cell responses to infection. This is currently under investigation.

In the present report, we found that early after vaccination the levels of circulation anti-S. enterica IgG antibodies were higher in FcγRI–/– FcγRII–/– FcγRIII–/– mice than in 129Ola/C57BL/6 control mice. This could be because phagocytes from mice lacking FcγR cannot bind anti-S. enterica antibodies and thus the usage of these bacterium-specific immunoglobulins is reduced during infection. It is also possible that the lack of the inhibitory FcγRII on B cells results in a higher production of IgG during vaccination. In fact, increased titres of antibacterial (e.g. Streptococcus pneumoniae) immunoglobulin have been described in vaccinated FcγRII–/– mice.52

Acknowledgments

This work was funded by a Medical Research Council grant to P. Mastroeni.

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