Abstract
Typhoid fever and nontyphoidal bacteremia caused by Salmonella remain critical human health problems. B cells are required for protective immunity to Salmonella, but the mechanism of protection remains unclear. In this study, we immunized wild-type, B cell–deficient, Ab-deficient, and class-switched Ab-deficient mice with attenuated Salmonella and examined protection against secondary infection. As expected, wild-type mice were protected and B cell–deficient mice succumbed to secondary infection. Interestingly, mice with B cells but lacking secreted Ab or class-switched Ab had little deficiency in resistance to Salmonella infection. The susceptibility of B cell–deficient mice correlated with marked reductions in CD4 T cell IFN-γ production after secondary infection. Taken together, these data suggest that the primary role of B cells in acquired immunity to Salmonella is via the development of protective T cell immunity.
Introduction
Typhoid fever is caused by infection with Salmonella typhi and is a serious health concern worldwide, causing an estimated 21 million cases and 216,000 deaths per year (1). Nontyphoidal salmonellosis (NTS) is caused by other Salmonella serovars and is a growing problem among HIV-infected adults and HIV-negative children in Africa and Asia (2–5). Currently, there are two vaccines for typhoid fever that each provide limited protection but are not widely used in endemic areas (6, 7). There is no available vaccine for NTS, although numerous target Ags have recently been defined (8). The development of novel, effective vaccines for typhoid and NTS requires greater understanding of Salmonella-specific T and B cell responses (9).
Immunity to Salmonella is studied using a well-established murine model of typhoid, in which Salmonella typhimurium causes fatal disseminated disease in susceptible, Nramps mice (10, 11). After oral infection, Salmonella can gain access to the mammalian host by invading M cells in the Peyer’s patches of the small intestine (10). Salmonella subsequently disseminates via the lymphatic system and replicates within phagocytic cells of the spleen, liver, and bone marrow. Salmonella actively inhibits phagolysosomal fusion, and infected macrophages require activation via IFN-γ to kill bacteria (12). Salmonella-specific Th1 cells that produce IFN-γ are essential for controlling bacterial growth, and mice lacking αβ CD4 T cells, Th1 cells, or IFN-γ eventually succumb to primary infection with attenuated bacteria (13, 14). Patients with primary genetic deficiencies in IL-12 or IFN-γ receptor signaling suffer from repeated disseminated Salmonella infections (15, 16). Thus, Th1 cells play an important role in mediating protective immunity in both human and murine salmonellosis.
The resolution of primary Salmonella infection confers robust protective immunity against secondary challenge. CD4 T cells are essential for this acquired resistance, and depletion of CD4 T cells eliminates the protective effect of vaccination with attenuated Salmonella (17). More surprisingly, for an intramacrophage infection, B cells are also essential for acquired immunity to Salmonella, and immunized B cell–deficient mice display enhanced susceptibility to secondary infection (18–20). However, the protective role of Abs in secondary immunity is somewhat controversial. Passive transfer of Abs is reported to be protective in some studies, whereas others have observed no protective effect (18, 19, 21). Furthermore, neither IgA nor mucosal Igs are required for protective immunity to Salmonella (8, 22). B cells can contribute to protective immunity via Ag presentation to Salmonella-specific Th1 cells (18, 23) or as an important source of inflammatory cytokines during infection (24, 25). However, it remains unclear whether the contribution of B cells to protective immunity is largely mediated by Ab-dependent or Ab-independent mechanisms.
In this study, we examined the role of B cells in protection against infection with virulent Salmonella using transgenic mouse strains that lack B cells, class-switched Ab, or Ab secretion and demonstrate that Ab production is largely dispensable for protection against secondary Salmonella infection. In contrast, B cells are required for optimal priming of Salmonella-specific Th1 cells that mediate bacterial clearance.
Materials and Methods
Mice
BALB/c (wild-type) and JhD/BALB/c (B cell–deficient) mice (National Cancer Institute, Frederick, MD) were used at 6–12 wk age. Transgenic membrane and secretory (m+s) IgM and membrane IgM (mIgM) use the B1–8 H chain, have a restricted BCR repertoire, were maintained on a JhD/BALB/c background (26). Transgenic mice were intercrossed with JhD/BALB/c mice and were used at 6–12 wk age. Homozygosity at the JHD locus was maintained by interbreeding with JhD mice, and PCR screening of the mIgM H chain was done using the following primers: Vh186.2 5′, CTACTGGATGCACTGGGTGA and Vh186.2 3′, TTGGCCCCAGTAGTCAAAGTA. All mice were housed in specific pathogen-free conditions for breeding and experimentation.
Bacteria and infection
Attenuated S. typhimurium BRD509 (ΔaroA/ΔaroD) and parental virulent strain SL1344 were grown overnight in Luria–Bertani broth and diluted in PBS after estimating bacterial counts by spectrophotometry. Mice were immunized i.v. with 5 × 105 BRD509 and challenged orally with 5 × 107 SL1344 after oral administration of 100 μl 5% NaHCO3. Infection doses were confirmed by plating serial dilutions onto MacConkey agar plates. Any moribund infected mice were euthanized as stipulated in our Institutional Animal Care and Use Committee protocol. Bacterial growth in vivo was calculated by plating serial dilutions of organ homogenates onto MacConkey agar, and bacterial counts were determined after overnight incubation at 37°C.
Detection of in vivo cytokine production and flow cytometry
Salmonella-specific CD4 and CD8 T cell responses were visualized as previously described (27). Immunized mice were injected i.v. with 1 × 108 heat-killed S. typhimurium
Salmonella-specific Ab response
Blood was collected by retro-orbital bleeding, and sera were prepared and stored at −20°C. Salmonella-specific IgM and IgG Abs were measured by ELISA, as previously described (27).
Statistical analysis
Statistical analysis was performed using unpaired t tests (Prism 4; GraphPad Software, La Jolla, CA). Survival data were compared using a log-rank (Mantel–Cox) test (Prism 4). Statistical differences between groups are delineated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.
Results and Discussion
Class-switched Abs are not required for secondary protection against Salmonella
Defining protective immune responses to Salmonella infection is a prerequisite for development of new effective vaccines against typhoid and NTS (10). Although CD4 T cells are critical for protective immunity to Salmonella, the contribution of B cells has not been clearly defined. Salmonella-specific Ab production, inflammatory cytokine production, and direct Ag presentation to T cells have each been proposed as mechanisms to explain the protective role of B cells during secondary infection (18, 19, 23, 24, 28). We sought to investigate whether B cells provide secondary protective immunity against Salmonella primarily in an Ab-dependent or -independent manner. Given previous data showing that serum transfer can protect against Salmonella (19), but that neither IgA nor mucosal Ig is required (8), we hypothesized that systemic IgG is essential for secondary clearance of bacteria. To test this hypothesis, we examined immunity in B cell–deficient mice (JhD), transgenic mice with B cells that cannot class switch or secrete Ab (mIgM), and mice with B cells that cannot class switch but are able to secrete IgM (m+s IgM) (26). Although the mIgM and m+s IgM transgenic mice have a restricted BCR repertoire, they do not have significant deviations in naive B cell and T cell subsets (Supplemental Fig. 1A–D and Ref. 29). All four strains (wild-type, B cell–deficient, mIgM, and m+s IgM mice) survived vaccination with attenuated S. typhimurium and had largely cleared bacteria from the spleen 44 d later (Supplemental Fig. 1E). This confirmed previous reports that resolution of primary infection with attenuated Salmonella does not require B cells (18, 19).
To examine acquired immunity to secondary Salmonella infection, naive and immunized mice from all four strains were challenged orally with virulent S. typhimurium (Fig. 1A). Regardless of the B cell compartment, all naive mice succumbed to primary infection with virulent Salmonella at a similar rate (Fig. 1A). In contrast, immunized wild-type mice resisted secondary infection with virulent Salmonella, whereas B cell–deficient mice succumbed to secondary challenge (Fig. 1A). Surprisingly, m+s IgM mice that lack class-switched Ab also survived secondary infection with Salmonella, demonstrating a similar degree of protective immunity to wild-type mice (Fig. 1A). Furthermore, most mIgM mice that lack all secreted Abs were resistant to secondary Salmonella infection. However, ∼25% of these mice eventually died of infection, and this was statistically different from the survival of wild-type and B cell–deficient mice (Fig. 1A). Taken together, these data confirm that B cells are essential for resistance to secondary infection with virulent Salmonella, and they surprisingly demonstrate that production of class-switched Abs is not required for protective immunity. Additionally, although secreted IgM Abs may contribute to secondary protection, the mechanism of B cell–mediated protection against secondary Salmonella infection is largely Ab-independent in this vaccination and rechallenge model.
Class-switched Abs are not necessary for immunity to Salmonella. (A) Naive wild-type, JhD, m+s IgM, and mIgM mice were infected orally with 5 × 107 S. typhimurium (SL1344) and survival was monitored. Wild-type, JhD, m+s IgM, and mIgM mice were immunized i.v. with 5 × 105 S. typhimurium (BRD509 ΔaroA/ΔaroD). Forty-two to 66 d later, mice were challenged orally with 5 × 107 S. typhimurium (SL1344) and survival was monitored. Data are pooled from three separate experiments and show the percentage of surviving mice in each group. The total number of mice is indicated. Survival of immunized wild-type, m+s IgM, and mIgM mice was statistically different (***p < 0.001) from JhD mice using a log-rank (Mantel–Cox) test. Survival of mIgM was also statistically different by log-rank test when compared with wild-type mice but not when compared with m+s IgM mice (*p < 0.05). (B) Mice were immunized i.v. with 5 × 105 BRD509, and at day 42 they were challenged orally with 5 × 107 SL1344 and serum was collected 9 d later. Data show levels of heat-killed Salmonella-specific IgM and IgG as determined by ELISA (n = 4–5 mice/group).
Given these findings, it was important to confirm the absence of circulating Salmonella-specific Ab in each B cell–deficient strain examined above. Serum was collected 9 d after secondary infection, and Salmonella-specific Ab responses were examined. Nine days after secondary infection, both wild-type mice and IgM Ab only (m+s IgM) mice had modest levels of circulating Salmonella-specific IgM (Fig. 1B), but only wild-type mice developed Salmonella-specific IgG (Fig. 1B). These results confirm that only wild-type mice produced a class-switched Ab response to Salmonella, but that IgM Ab only mice developed low Salmonella-specific IgM responses during secondary infection.
Secondary bacterial clearance does not require class-switched Abs
Given the fact that mice lacking all Abs had a 25% death rate following virulent challenge, it seemed likely that bacterial clearance was hindered at late time points in these mice, perhaps because IgM is required for clearance from a particularly persistent anatomical site such as the mesenteric lymph nodes (30). Thus, we examined the rate of bacterial clearance in immunized mice lacking B cells, class-switched Abs, or all Abs. Three days after secondary infection, wild-type mice had lower bacterial loads in the spleen than did B cell–deficient mice (Fig. 2A), demonstrating that B cells are required for rapid secondary clearance of bacteria. At this early time point, no significant differences were apparent between Ab-deficient strains and B cell–deficient mice, but Ab-deficient mice had a trend toward lower CFUs in the spleen (Fig. 2A). No significant differences were detected in liver CFUs at this same early time point (Fig. 2B). Nine days after secondary infection, mice lacking B cells had much higher bacterial loads in both the spleen and liver compared with wild-type mice (Fig. 2). In marked contrast, mIgM and m+s IgM mice had lower CFUs in both spleen and liver (Fig. 2). Taken together, these data demonstrate that the rate of bacterial clearance during secondary infection is largely unaffected by the absence of Abs, despite a requirement for B cells. This finding contrasts with prior studies that showed a protective effect of serum transfer (19, 21). However, these studies were not designed to test an Ab-independent role of B cells, and both studies described protection against low dose challenge. Our finding has broad implications because the measurement of circulating Ig is often used as an indicator of vaccine efficacy.
Rapid bacterial clearance does not require Abs. (A and B) Mice were immunized i.v. with 5 × 105 BRD509 and 42–66 d later challenged orally with 5 × 107 SL1344. Three and 9 d later bacterial counts were determined in (A) spleen and (B) liver. Data show mean log10 CFUs per organ (n = 6–26 mice per group/time point; *p < 0.05, **p < 0.01, ***p < 0.001).
B cell–deficient mice have reduced CD4 T cell responses to Salmonella
It is clear from previous work that secretion of IFN-γ by Th1 cells is critical for the resolution of Salmonella infections (14, 31). We confirmed this by depleting CD4 and CD8 T cells in immunized wild-type mice and challenging them with a virulent strain of Salmonella. T cell depletion caused a significant increase in bacterial loads during secondary infection (Supplemental Fig. 2A). It has been suggested that Abs can enhance T cell responses to Salmonella by allowing bacterial uptake via Fc receptors on dendritic cells (32). B cells also can present Ag and secrete cytokines that shape the development of protective T cell responses. Thus, we examined the effect of B cell or Ab deficiency on the generation of Salmonella-specific Th1 cells.
Wild-type, B cell–deficient, m+s IgM, and mIgM mice were immunized with attenuated Salmonella, and Salmonella-specific CD4 T cell responses were examined 42 d later. As previously reported (33, 34), immunized wild-type mice had a large population of CD4 T cells that produced IFN-γ in response to HKST stimulation (Fig. 3A, Supplemental Fig. 2B). In marked contrast, immunized B cell–deficient mice had lower numbers of IFN-γ–producing Th1 cells in response to HKST (Fig. 3A, Supplemental Fig. 2B). This difference was Ab-independent, as immunized m+s IgM and mIgM mice had similar levels of IFN-γ–producing CD4 T cells as did wild-type mice (Fig. 3A, Supplemental Fig. 2B). In fact, mIgM mice, which lack all secreted Abs, had a larger population of Salmonella-specific IFN-γ–producing CD4 T cells. Interestingly, IFN-γ–producing CD8 T cells were also slightly reduced in immunized B cell–deficient mice, but this was not statistically significant (Fig. 3B, Supplemental Fig. 2B). Taken together, these data indicate that B cells, but not Abs, are required for shaping the development of protective CD4 Th1 responses to Salmonella. A similar role for B cells has been reported in other infection models such as lymphocytic choriomeningitis virus and Pneumocystis (35, 36). Although B cells may directly present Ag and drive Salmonella-specific Th1 responses, a recent study demonstrated that B cell production of IL-6 is important for maximal Th17 responses, and B cell production of IFN-γ contributed to Th1 development (24). A recent study has also shown that B cells can negatively affect secondary responses to Salmonella infection via an MyD88- and IL-10–dependent mechanism (37). Thus, B cells likely contribute to protective CD4 responses via Ag presentation and production of specific cytokines that drive effector lineage commitment during primary responses. It is not yet clear whether these required B cells are necessarily Salmonella-specific, but the limited B cell repertoire in IgM only mice and in no Ab mice did not affect protective immunity. We also attempted to address this issue using in vitro restimulation and B cell tetramer pull-down experiments in previously infected mice, but we did not detect an elevated frequency of Salmonella-specific B cells using either of these approaches. However, it remains possible that expanded Salmonella-specific B cells contribute to immunity to secondary infection.
Ab is not required for optimal Salmonella-specific Th1 cells. (A and B) Mice were immunized i.v. with 5 × 105 BRD509 and 42–47 d later injected i.v. with 108 HKST to stimulate T cell responses. Bar graphs showing mean number of (A) CD4 or (B) CD8 T cells producing IFN-γ after stimulation with HKST (**p < 0.01, ***p < 0.001).
Collectively, our data demonstrate that Ab production plays only a minor role in Salmonella immunity in our live vaccination model, whereas B cells are required for the development of protective T cell immunity. These findings will be important for the development of new effective vaccines against typhoid and NTS.
Disclosures
The authors have no financial conflicts of interest.
Footnotes
This work was supported by National Institutes of Health Grants AI091298 (to M.R.N.), AI087830 (to S.S.W.), AI043603 (to M.J.S.), AI055743 and AI073672 (to S.J.M.), and T32 GM008244 (to the University of Minnesota Medical Scientist Training Program).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- HKST
- heat-killed Salmonella typhimurium
- mIgM
- membrane IgM
- m+s IgM
- membrane and secretory IgM
- NTS
- nontyphoidal salmonellosis.
- Received May 25, 2012.
- Accepted October 18, 2012.
- Copyright © 2012 by The American Association of Immunologists, Inc.