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Originally published online as doi:10.1189/jlb.0306194 on August 17, 2006

Published online before print August 17, 2006
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(Journal of Leukocyte Biology. 2007;81:403-411.)
© 2007 by Society for Leukocyte Biology

Exposure to LPS suppresses CD4+ T cell cytokine production in Salmonella-infected mice and exacerbates murine typhoid

Aparna Srinivasan and Stephen J. McSorley1

Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, and Center for Infectious Diseases and Translational Microbiology Research, University of Minnesota Medical School, McGuire Translational Research Facility, Minneapolis, Minnesota, USA

1 Correspondence: Dept. of Medicine, Div. of Gastroenterology, Hepatology and Nutrition, and Center for Infectious Diseases and Translational Microbiology Research, University of Minnesota Medical School, McGuire Translational Research Facility, Room 3-4006, 2001 6th St., SE, Minneapolis, MN 55455, USA. E-mail: mcsor002{at}umn.edu

ABSTRACT

A number of studies have documented suppression of lymphocyte activation in mice infected with Salmonella. Here, we describe incomplete activation of CD4+ T cells following intravenous injection of specific peptide and LPS into Salmonella-infected mice. Although antigen-specific CD4+ T cells were activated by peptide/LPS to increase surface CD69 expression, they did not produce IL-2 or TNF-{alpha}. Suppression of cytokine production did not require prolonged exposure of the T cells to the Salmonella-infected environment, was not antigen specific, but was dependent upon the presence of LPS during stimulation. These data suggest that Salmonella-infected mice are exquisitely sensitive to the generation of a suppressive environment following innate immune stimulation with LPS. In agreement with this interpretation, repeated low-dose administration of LPS caused uncontrolled replication of attenuated Salmonella in vivo.

Key Words: bacterial infection • IL-2 • tumor necrosis factor-{alpha}

INTRODUCTION

Typhoid fever, caused by infection with Salmonella enterica serovar typhi, has a profound detrimental effect on the health care and economy of many developing nations. The development of improved typhoid vaccines remains a high priority, especially those that could be targeted to young children and the elderly living in endemic areas.

The immune response to typhoid has been widely studied using a mouse model of infection with Salmonella enterica serovar typhimurium [1 , 2 ]. Although there are some differences between murine and human disease, it remains the best available and most widely used model to understand protective immunity during typhoid [3 ]. Attenuated strains of Salmonella cause a transient infection in mice and induce protective immunity against future exposure to virulent strains of Salmonella [4 ]. CD4+ T cells are essential for this vaccine-induced protective immunity [5 , 6 ], although CD8 T cells and antibody responses can also contribute [7 8 9 10 ]. The specific immune targets recognized by protective CD4+ T cells are mostly undefined [11 ], although class-II epitopes in bacterial flagellin and SipC have been reported [12 13 14 ].

About 20 years ago, the phenomenon of Salmonella-induced immune suppression was first described [15 , 16 ]. Eisenstein et al. [15 , 16 ] noted that spleen cells recovered from mice infected with attenuated Salmonella were profoundly unresponsive to B and T cell mitogens in vitro. In subsequent studies, this suppression was attributed to the presence of suppressive macrophages that inhibit lymphocyte activation through the production of nitric oxide [17 , 18 ]. In vitro immune suppression of infected spleen cell cultures was somewhat puzzling because infected mice can actively clear bacteria in vivo and are simultaneously resistant to cross-challenge with other bacteria [19 ]. This phenomenon has therefore been referred to as "the paradox of Salmonella immunity and immunosuppression" [20 , 21 ]. To complicate matters further, inhibition of nitric oxide production eliminates in vitro immune suppression of T cell responses, but also prevents the clearance of bacteria in vivo [22 ], emphasizing the dual role of this mediator. Although Salmonella-induced immune suppression has been clearly documented in vitro, it remains unclear whether this phenomenon actually occurs in vivo, and if so, how it may influence the induction of protective immunity to Salmonella.

Our laboratory has actively studied Salmonella flagellin-specific CD4+ T cell activation in vivo using a TCR transgenic adoptive transfer approach [23 , 24 ]. Flagellin-specific CD4+ T cells are rapidly activated after oral or IV infection with virulent or attenuated Salmonella [23 , 24 ], and therefore provide a useful model to track Salmonella-specific T cell activation in vivo [25 ]. Here, we show that CD4+ T cells in Salmonella-infected mice have reduced ability to produce IL-2 or TNF-{alpha} when activated by injection of specific peptides and LPS. However, this immune suppression is not an intrinsic feature of T cells in Salmonella-infected mice but is acquired by stimulation in the presence of LPS. Furthermore, administration of LPS to Salmonella-infected mice caused uncontrolled bacterial replication in vivo. These data suggest that CD4+ T cells are not actually suppressed in Salmonella-infected mice but can rapidly become suppressed when exposed to bacterial adjuvants.

MATERIALS AND METHODS

Mouse strains
SM1 Rag-deficient mice express a transgenic rearranged Vß2V{alpha}10 T cell receptor that confers reactivity to a peptide from Salmonella flagellin (427-441) [13 ]. SM1 mice expressing either CD90.1 or CD45.1 alleles have previously been described [24 ]. OT-II TCR transgenic cells were a kind gift from Dr. Marc Jenkins, University of Minnesota. Rag-2-deficient C57BL/6 mice were bred at the University of Minnesota. C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and used at 8–16 weeks of age. All mice were housed in specific pathogen-free conditions and cared for in accordance with institutional guidelines.

Adoptive Transfer of TCR transgenic T cells
Spleen and lymph node cells (inguinal, axillary, brachial, cervical, mesenteric, and periaortic) were harvested from SM1 RAG-deficient or OT-II RAG-deficient TCR transgenic mice, and a single-cell suspension was generated. In some experiments, CD4 cells from C57BL/6 and SM1 RAG-deficient mice were stained with antibodies to CD4, Vß2, CD11a, CD25, CD44, CD62L, CD69, and CD127 to determine the percentage of naïve and memory T cells. For adoptive transfer experiments an aliquot of SM1 cells was stained using antibodies to CD4+, CD90.1, (or CD45.1+) and Vß2 (BD Biosciences, San Diego, CA) while OT-II cells were stained with antibodies to CD4+, and Vß6. The percentage of TCR transgenic cells was assessed by flow cytometry using a FACSCalibur (Becton-Dickenson, Mountain View, CA). Cell numbers were adjusted accordingly and 2-5x106 TCR transgenic T cells were injected intravenously into recipient C57BL/6 mice. In most experiments, TCR transgenic cells were also stained with the dye CFSE [26 ] immediately before adoptive transfer.

Salmonella infection
Salmonella enterica serovar typhimurium strains, SL1344 or AroA–D– BRD509 were grown overnight in LB broth without shaking, diluted in PBS after an estimation of bacterial concentration using a spectrophotometer, and injected into the lateral tail vein of recipient mice. In all infection experiments the dose of bacteria administered was confirmed by plating serial dilutions on MacConkey agar plates.

T cell stimulation in vivo
Groups of adoptively transferred mice were injected intravenously with 200µg of flagellin peptide [13 ], or OVA peptide, plus 25µg of E. coli LPS L4391 (Sigma, St. Louis, MO). In some experiments, mice were injected with peptide without LPS or with graded concentrations of LPS. To examine cytokine production directly ex vivo, spleens from injected mice were harvested 6 h after peptide injection. Cells were immediately surface stained at 4°C, fixed with formaldehyde, permeabilized using saponin (Sigma), and stained intracellularly using anti-cytokine antibodies or isotype controls.

Flow cytometric analysis
Spleen cells were incubated on ice for 20-45 min in Fc block (spent culture supernatant from the 24G2 hybridoma, 2% rat serum, 2% mouse serum, and 0.01% sodium azide) in the presence of the relevant primary antibodies. Fluoroscein isothiocyanate- (FITC), phycoerytherin- (PE), CyChrome-, PE-Cy5-, or allophycocyanin-conjugated antibodies specific for CD4+, CD45.1+, CD69, CD90.1, IL-2, and TNF-{alpha} were purchased from PharMingen. After staining, cells were analyzed by flow cytometry using a FACSCalibur. Data were analyzed using FlowJo software (TreeStar, San-Carlos, CA).

Evaluating bacterial load in vivo
At various times after infection, spleen cells were harvested in PBS and serial dilutions of each sample were plated onto MacConkey agar plates (Difco, Detroit, MI) to determine bacterial colonization. Plates were incubated overnight at 37°C and bacterial colonies were counted the following day.

RESULTS

SM1 T cells do not respond to low-dose infection and are unable to respond to stimulation with peptide/LPS
We recently reported an adoptive transfer system using SM1 TCR transgenic T cells specific for Salmonella flagellin [23 ]. While spleen cells from C57BL/6 mice contain populations of naïve and memory CD4 T cells, in contrast, SM1 CD4 T cells recovered from donor mice express a phenotypically naïve pattern of surface markers; CD11alo, CD25–, CD44lo, CD62LHi, CD69, CD127Hi (Fig. 1A ). Thus, this TCR transgenic mouse model allows us to examine the activation of naïve Salmonella-specific T cells in vivo.


Figure 1
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  		Figure 1. SM1 T cells expand in response to high-dose, but not low-dose Salmonella infection. (A) CD4 T cells were recovered from the spleen of Rag-deficient SM1 transgenic and C57BL/6 mice and immediately stained with antibodies specific for CD4, Vß2, CD11a, CD25, CD44, CD62L, CD69, and CD127. Plots show surface staining after gating of CD4 T cells. (B) C57BL/6 mice were adoptively transferred with 2 x 106 CFSE-stained SM1 T cells and infected intravenously the following day with 103 (low dose) or 105 (high dose) Salmonella, SL1344. Three days later, the percentage of SM1 T cells and CFSE-dye dilution was examined in the spleen of uninfected (transfer only), or infected mice. Boxes and numbers show the percentage of SM1 T cells as a percentage of all spleen cells. CFSE plots (lower) show FL1 fluorescence after gating on SM1 cells using boxed gates similar to those presented in upper panels. Plots are similar to 3 mice per group and 2 separate experiments.

 
We have previously reported that SM1 T cells respond vigorously to high-dose Salmonella infection but do not respond to low-dose infection [24 ]. This is most likely due to the modulation of flagellin expression in vivo and/or antigen sequestration away from dendritic cells in infected lymphoid tissues [24 , 27 , 28 ]. Groups of mice were adoptively transferred with SM1 T cells and infected the following day with 1 x 105, or 1 x 103 Salmonella. Three days later, the percentage of SM1 T cells in the spleen of high-dose infected mice had expanded ~10-fold, and almost all of the cells had undergone several rounds of cell division (Fig. 1B ; high-dose infection). In contrast, the percentage of SM1 T cells was similar in uninfected and low-dose infected mice, and SM1 cells displayed no evidence of cell division (Fig. 1B) . We have previously reported that these unresponsive SM1 T cells display no evidence of T cell activation by examination of surface marker expression [24 ] and are therefore most likely naïve cells that have not encountered antigen in vivo.

Numerous reports have described Salmonella-induced immunosuppression of T cell responses [20 , 21 ]. This phenomenon has been examined extensively in vitro but has not been examined in vivo using a TCR transgenic approach. The unresponsiveness of SM1 T cells after low-dose infection provided an opportunity to examine whether Salmonella-induced immune suppression could be observed in vivo. Mice were adoptively transferred with CFSE-stained SM1 T cells, infected with a low dose (103) of Salmonella for 3 days, and then T cells activated in vivo by intravenous injection of specific peptide and LPS. This method of in vivo stimulus was used because it has been shown to cause rapid and synchronous activation of transgenic T cells in vivo [29 ]. At this time point, infected mice were found to have a mean of 925,000 ± 106,066 bacteria per spleen yet SM1 T cells displayed little evidence of CFSE-dye dilution in response to Salmonella infection (Fig. 2A , right). SM1 T cells in the spleen of uninfected (transfer only) mice did not express CD69 on the cell surface or produce detectable IL-2 (Fig. 2A , left). However, 6 h after injection of peptide/LPS, most SM1 cells expressed high levels of CD69 and a large proportion of cells produced IL-2 (Fig. 2A , middle). In marked contrast, SM1 T cells in Salmonella-infected mice did not produce IL-2 in response to peptide/LPS injection (Fig. 2A , bottom right). This was not due to a defect in TCR ligation, as most SM1 T cells increased surface levels of CD69 in response to the stimulus (Fig. 2A) . Thus, SM1 T cells in Salmonella-infected mice are activated by peptide/LPS to increase CD69 expression but do not produce IL-2. Furthermore, infected mice administered peptide/LPS died more rapidly, within 2 days of injection (data not shown). Naïve SM1 T cells do not produce effector cytokines such as IFN-{gamma} at this early time point (data not shown)


Figure 2
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  		Figure 2. Suppression of SM1 cytokine production in Salmonella-infected mice. C57BL/6 mice were adoptively transferred with 2 x 106 CFSE-labeled SM1 T cells and infected intravenously the following day with 103 Salmonella, SL1344. (A) Three days later, groups of uninfected or infected mice were injected intravenously with 200-µg flagellin peptide plus 25 µg LPS and surface CD69 expression, and intracellular IL-2 production by SM1 T cells was examined 6 h later. Plots show CFSE, CD69, and IL-2 staining after gating on SM1 T cells by CD4 and CD90.1 surface staining. Individual plots are similar to 3 mice per group and 2 experiments. (B) At 24-h intervals after infection, groups of infected mice were injected intravenously with 200 µg flagellin peptide plus 25µg LPS and surface CD69 expression, intracellular IL-2 and TNF-{alpha} production was examined 6 h later. Plots show the mean percentage of positive SM1 cells ± SD, after gating on CD4+CD90.1+ cells. Each data point is representative of 3 mice per time point.

 
We next determined how quickly this suppression of T cell cytokine production developed after Salmonella infection. SM1 T cells were stimulated by peptide/LPS injection at 24-h intervals over the first 3 days of low-dose (103) Salmonella infection. No defect in SM1 production of IL-2 or TNF-{alpha} was detected on the day of infection, or 1 day after infection (Fig. 2B) . However, 2 days after Salmonella infection SM1 T cells had developed a defect in the production of IL-2 and TNF-{alpha}, which became more pronounced the following day (Fig. 2B) . In marked contrast, increased expression of CD69 remained unaffected at all time points after Salmonella infection (Fig. 2B)

Short exposure to the Salmonella-infected environment is sufficient to cause suppression of T cell cytokine production
The previous experiment demonstrated that suppression of cytokine production was first observed 2 days following Salmonella infection. However, it was unclear whether T cells required a full 2 days in the infected host to experience cytokine inhibition, or whether it merely took 2 days to generate a suppressive environment that had immediate impact on T cell responses. We therefore examined whether a freshly transferred SM1 population would experience the same level of cytokine inhibition as an identical population that had been present in the mouse for 3 days. We previously generated 2 SM1 mouse strains with identical specificity but differing in expression of CD45.1 or CD90.1 [24 ]. C57BL/6 mice were adoptively transferred with CD90.1+ SM1 T cells and infected the following day with 103 Salmonella. Three days later, the same mice were adoptively transferred with fresh CD45.1+ SM1 T cells and both populations were stimulated by injection of peptide/LPS. This allowed comparison of cytokine production from SM1 T cells that had been circulating in the infected host for 3 days (day 3 cells) or only a few hours (new cells). Both populations of SM1 T cells could be clearly distinguished from endogenous CD4+ cells by expression of CD90.1(day 3) or CD4+5.1(new) (Fig. 3 -gated populations). In adoptively transferred, but uninfected mice, both SM1 T cell populations (new and day 3) produced IL-2 and TNF-{alpha} in response to peptide/LPS stimulation (Fig. 3 , left panels). However, peptide-specific IL-2 and TNF-{alpha} production by both SM1 populations (new and day 3) was greatly reduced in infected mice (Fig. 3 , right). Therefore, short exposure of SM1 T cells to the Salmonella-infected environment is sufficient to induce suppression of cytokine responses.


Figure 3
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  		Figure 3. Immune suppression requires only short exposure to the infected environment. C57BL/6 mice were adoptively transferred with 2 x 106 CD90.1+ SM1 T cells (day 3) and infected intravenously the following day with 103 Salmonella, SL1344. Three days later mice were adoptively transferred with 2 x 106 CD45.1+ SM1 T cells (new) and 6 h later, groups of uninfected or infected mice were injected intravenously with 200 µg flagellin peptide plus 25 µg LPS (+ peptide) or left untreated (– peptide). Six hours later, spleens were harvested and CD90.1+ (day 3) and CD45.1+ (new) SM1 T cells detected by surface staining (upper plots). Intracellular IL-2 and TNF-{alpha} production in response to peptide stimulation by each population of SM1 T cells is presented. Individual plots are similar to 3 mice per group and 2 separate experiments.

 
Salmonella-induced suppression of bystander T cell cytokine production
We next examined whether the suppressive effect of Salmonella infection was dependent upon TCR expression or whether any T cell response would be suppressed. Given the specificity of SM1 T cells for Salmonella flagellin, it seemed possible that inhibition of cytokine production could be related in some way to this particular antigen specificity. Mice were adoptively transferred with CD90.1+ SM1 T cells and a population of CFSE-labeled, Ovalbumin-specific OT-II T cells before infection with 103 Salmonella. Three days after infection, groups of mice were injected with flagellin peptide/LPS or OVA peptide/LPS to elicit cytokine production in vivo. Injection of flagellin peptide/LPS induced CD69 expression on the surface of most SM1 T cells in infected and uninfected mice (Fig. 4 ). As described above, SM1 T cells in Salmonella-infected mice produced much less IL-2 or TNF-{alpha} in response to stimulation compared with uninfected mice (Fig. 4) . Similarly, most OT-II cells in uninfected and Salmonella-infected mice increased expression of CD69 in response to OVA peptide/LPS stimulation. However, IL-2 and TNF-{alpha} production by OT-II cells were suppressed in Salmonella-infected mice when compared with uninfected mice (Fig. 4) . Therefore, Salmonella infection causes suppression of cytokine production from CD4+ T cells irrespective of antigen specificity.


Figure 4
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  		Figure 4. Salmonella-induced suppression of bystander OT-II T cells. C57BL/6 mice were adoptively transferred with 2 x 106 SM1 T cells and 2 x 106 CFSE-labeled OT-II T cells and infected intravenously the following day with 103 Salmonella, SL1344. Three days later, mice were injected intravenously with 200 µg peptide plus 25 µg LPS (+ peptide/LPS) and 6 h later, spleens were harvested. SM1 T cells were stimulated by injection of flagellin peptide plus LPS, whereas OT-II T cells were stimulated by OVA peptide plus LPS. Surface CD69 expression, intracellular IL-2, and TNF-{alpha} production were examined after gating on CD4+CFSEHI (OT-II) or CD4+CD90.1+ (SM1) T cells. Plots show the mean percentage of positive OT-II or SM1 cells ± SD, and each bar is representative of 3 mice per time point.

 
Suppression of cytokine responses is dependent upon the presence of LPS in activation stimulation
As noted above, peptide/LPS was reported as an ideal stimulus for the simultaneous activation of TCR transgenic T cells in vivo [29 ]. However, TCR transgenic cells will produce lower levels of cytokines in response to peptide injection alone. Therefore, we examined whether inclusion of LPS as part of the activation stimulus was involved in the establishment of immune suppression in vivo. Mice were adoptively transferred with SM1 T cells and infected the following day with 103 Salmonella. Three days later, mice were injected with flagellin peptide without LPS, and SM1 T cell activation was examined. SM1 T cells in both uninfected (transfer only) and infected mice increased surface expression of CD69 in response to peptide (Fig. 5 , top). Surprisingly, SM1 T cells in uninfected and infected mice also produced similar amounts of IL-2 (Fig. 5 , bottom). These data suggested that LPS injection, as part of the TCR transgenic T cell stimulus, was actually responsible for inducing immune suppression in Salmonella-infected mice.


Figure 5
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  		Figure 5. SM1 T cells in infected mice produce IL-2 after activation with peptide alone. C57BL/6 mice were adoptively transferred with 2 x 106 SM1 T cells, and groups of mice were infected intravenously with 103 Salmonella (SL1344). Three days later, some mice were immunized intravenously with 200 µg flagellin peptide (+ peptide), and others left untreated. (A) Six hours later, spleen cells were harvested and surface stained using monoclonal antibodies against CD4+, CD90.1, and CD69. Cells were then fixed and stained for intracellular IL-2 production. Plots show CD69 and IL-2 staining after gating on SM1 (CD4+, CD90.1+) T cells and are representative of 2 individual experiments.

 
To confirm this finding, we also examined SM1 T cell proliferation in vivo, in the presence or absence of LPS. Groups of mice were adoptively transferred with SM1 T cells and infected the following day with 103 Salmonella. Three days later, mice were injected with LPS alone, peptide alone, or peptide plus graded concentrations of LPS to stimulate T cell activation. Six hours later SM1 T cells had increased expression of CD69 in all peptide and peptide plus LPS groups, indicating activation had occurred (data not shown). Two days later, SM1 T cells in uninfected and Salmonella-infected mice had expanded and diluted CFSE in response to peptide injection without LPS (Fig. 6 , peptide alone, and data not shown). In fact, peptide injection of Salmonella-infected mice induced greater SM1 proliferation than was observed in uninfected mice (Fig. 6) . As expected, coinjection of uninfected mice with increasing concentrations of LPS with peptide increased the extent of SM1 T cell expansion (Fig. 6 , left). In marked contrast, injection of increasing amounts of LPS actually decreased SM1 T cell proliferation in Salmonella-infected mice (Fig. 6 , right). These data demonstrate that there is no intrinsic suppression of T cell activation in Salmonella-infected mice but that injection of LPS can cause suppression of T cell responses in vivo.


Figure 6
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  		Figure 6. Stimulation of SM1 T cells in the presence of LPS inhibits T cell proliferation. C57BL/6 mice were adoptively transferred with 2 x 106 SM1 T cells, and groups of mice were infected intravenously with 103 Salmonella (SL1344). Three days later, some mice were immunized intravenously with 25 µg of LPS (No peptide/LPS), 200 µg flagellin peptide (peptide alone), or peptide with graded concentrations of LPS. Two days later, spleen cells were harvested and the percentage of CD4+ CD90.1+ SM1 T cells was determined. Numbers above the boxed gate represent the percentage of SM1 T cells in the spleen of each mouse. ({dagger}) indicates that Salmonella-infected mice injected with peptide+25 µg of LPS had died and therefore could not be included in the analysis.

 
LPS administration to infected mice causes uncontrolled bacterial growth
In the previous experiment, we were unable to examine the extent of SM1 T proliferation in response to peptide + 25 µg LPS since all mice died after LPS injection and prior to analysis, 2 days later (Fig. 6 , {dagger}). Together, our data suggested that LPS administration can induce an environment in which T cell responses are inhibited. Therefore, we examined the effect of LPS administration upon growth of attenuated Salmonella in vivo. We used the bacterial strain BRD509 that has deletions in aroA and aroD genes and is therefore attenuated in C57BL/6 mice [30 ]. Mice were infected i.v. with 105 BRD509 and one group of mice received six injections of 5 µg of LPS on days 1, 2, 3, 4, 5, and 6 postinfection. Bacterial numbers were examined in the spleen at days 7, 14, and 21-postinfection. In LPS-untreated infected mice, numbers of BRD509 reached a peak 14 days after infection but were clearly reduced by day 21 (Fig. 7A ), constant with immune control of bacterial growth, as described previously [30 , 31 ]. However, in mice administered LPS, bacterial numbers were much higher at day 14 and remarkably showed no evidence of control by day 21 after infection. These data demonstrate that low-dose LPS administration has a profoundly detrimental effect upon immune clearance of attenuated Salmonella.


Figure 7
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  		Figure 7. Administration of LPS causes uncontrolled growth of attenuated Salmonella. (A) C57BL/6 mice were infected intravenously with 105 Salmonella, BRD509 and one group of mice received daily intravenous injections of 5 µg of LPS for the first 6 days of infection. Spleens were removed at days 7, 14, and 21 and the number of Salmonella determined. Data points show mean number of bacteria ± SD for 3 mice per time point. (B) C57BL/6 or Rag-deficient C57BL/6 mice were infected intravenously with 105 Salmonella, BRD509 and some mice were given daily intravenous injections of 5 µg of LPS for the first 6 days of infection. Spleens were removed at day 20, and the number of bacteria was determined. Data points show mean number of bacteria ± SD for 3 mice per time point.

 
Next, we sought to determine whether LPS treatment caused higher bacterial loads as a result of the suppression of lymphocyte responses or through LPS effects on nonlymphocytes. C57BL/6 or C57BL/6 Rag-deficient mice were infected i.v. with 105 BRD509, and groups of mice were injected with 5 µg of LPS on days 1, 2, 3, 4, 5, and 6 postinfection. Bacterial numbers were examined in the spleen 20 days after infection and, as expected, bacterial burdens were slightly higher in Rag-deficient compared with C57BL/6 mice (Fig. 7B) . LPS administration increased bacterial burdens in both groups of mice, demonstrating that a portion of the LPS-suppressive effect is due to effects on nonlymphocytes. However, bacterial loads were much higher in LPS-treated C57BL/6 mice vs. LPS-treated Rag-deficient mice, indicating that the suppressive effect of LPS on lymphocyte responses can influence bacterial growth in vivo.

DISCUSSION

A number of reports have described the inhibition of CD4+ T cell and B cell responses in cultures of spleen cells harvested from Salmonella-infected mice [20 , 21 ]. This phenomenon has been examined in some detail, and the suppressive effect attributed to the production of nitric oxide by macrophages in vitro [17 , 18 ]. This suppression is surprising as it correlates with an ongoing protective immune response against Salmonella in vivo, and cross-protection against secondary Listeria infection [19 ]. However, although suppression of immune responses by Salmonella has been reported in vitro, it has not been observed in vivo. Our data demonstrate that suppression of T cell responses can occur in vivo in Salmonella-infected mice, that this effect is not antigen-specific, and does not require prolonged T cell exposure to the infected environment. Although there are obvious differences between the in vivo assay system used in our study and the in vitro assays employed by previous investigators [20 , 21 ], it seems likely that the same phenomenon is being observed in both cases. However, it should be noted that previous work has focused on the role of macrophage nitric oxide production, whereas our in vivo assays have not examined the mechanism of T cell suppression in vivo. Together, these data reinforce the potential for suppression of lymphocyte responses during murine, and possibly human typhoid.

One unexpected finding of our study was that administration of LPS was required to induce suppression of T cell responses in Salmonella-infected mice. This result implies that immune suppression is not a constitutive feature of murine typhoid but that infected mice have a propensity to generate a suppressive environment following exposure to a bacterial adjuvant. Previous in vitro stimulation studies examining immune suppression did not require addition of LPS or other bacterial adjuvants to observe immune suppression [20 , 21 ]. However, it is possible that these experiments may have inadvertently exposed Salmonella-infected spleen cells to bacterial adjuvants via the mechanical disruption of Salmonella-infected tissue or even by exposure to trace endotoxin in tissue culture plastic. If this is true, then our data would clarify the previously noted "paradox" of immune suppression and immunity in murine typhoid [20 , 21 ], by suggesting that immune suppression does not normally occur without exposure to further stimulation in vitro or in vivo. Interestingly, many of the initial experiments describing in vitro immune suppression were carried out using LPS-unresponsive mouse strains [15 ], suggesting that LPS was not responsible for activation of spleen cells in these experiments. However, many other bacterial products, including CpG DNA and flagellin have a similar ability to activate innate immune responses [32 , 33 ] and may therefore have contributed to in vitro activation in previous experiments. Of course, it remains possible that our data and previous studies by Eisenstein et al. [15 , 16 , 20 ] have examined separate and distinct immune suppression phenomena, both of which may occur during typhoid.

Administration of low doses of LPS had a detrimental effect on control of bacterial growth in vivo (Fig. 7) . These data are reminiscent of the synergistic lethality reported between viral infection and bacterial superinfection in mouse models of sepsis [34 ]. Indeed, viral infection also predisposes mice to succumb to an otherwise nonlethal injection of LPS [35 , 36 ]. Although susceptible mice normally resolve infection with attenuated strains of Salmonella, it is known that this is contingent upon the development of an active immune response. Thus, mice deficient in CD4+ T cells or the production of IFN-{gamma} are profoundly susceptible to infection with attenuated Salmonella [37 , 38 ]. From our study of Rag-deficient vs. C57BL/6 mice, it is clear that LPS administration can impede both lymphocyte-, and nonlymphocyte-mediated bacterial clearance in vivo. It seems most likely that part of the effect of LPS administration involves the inhibition of naïve T cell activation or the effector functions of Salmonella-specific CD4+ T cells in vivo. One interesting note from our study is that LPS encouraged the growth of bacteria between 14 and 21 days after infection, yet was only administered during the first week of infection. These data underline the importance of LPS-induced immune suppression upon Salmonella infection and suggest that brief exposure to LPS can have long-term effects on bacterial control.

Our in vivo T cell stimulation assays used E. coli LPS rather than Salmonella LPS. Purified Salmonella LPS has the potential to be contaminated by Salmonella flagellin, the antigenic target of our SM1 T cells, thus complicating the interpretation of any stimulation experiments. In contrast, we previously demonstrated that SM1 T cells do not respond to E. coli flagellin [13 ], allowing us to avoid possible contamination with a flagellin that would activate SM1 T cells.

What is the physiological importance for LPS-induced immune suppression in human or murine typhoid? Because immune suppression was only observed in Salmonella-infected mice following further exposure to LPS, these data argue that suppression is not a consistent feature of murine typhoid in the laboratory. However, many patients suffering from typhoid reside in endemic areas where repeated exposure to bacterial LPS would be very likely to occur. Exposure to LPS, from Salmonella or other pathogens, may impair ongoing host defense to the primary infection and exacerbate the disease in typhoid patients. Few studies have examined the impact of secondary infection upon immune response to typhoid, but it seems possible that immune suppression as a result of secondary exposure could contribute significantly to the pathogenesis of typhoid. Future studies are required to examine this important issue.

ACKNOWLEDGEMENTS

We would like to thank Joe Foley for mouse breeding and technical assistance. This work was supported by grants from National Institutes of Health (AI056172 and AI055743 to S. J. M).

Received March 13, 2006; revised April 18, 2006; accepted May 9, 2006.

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