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The Journal of Immunology, 2007, 178, 6342-6349
Copyright © 2007 by The American Association of Immunologists, Inc.
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Innate Immune Activation of CD4 T Cells in Salmonella-Infected Mice Is Dependent on IL-181

Aparna Srinivasan*, Rosa-Maria Salazar-Gonzalez*, Michael Jarcho*, Michelle M. Sandau{dagger}, Leo Lefrancois{ddagger} and Stephen J. McSorley2,*

* Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, and Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, McGuire Translational Research Facility, Minneapolis, MN 55455; {dagger} Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN 55455; and {ddagger} Division of Immunology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Production of IFN-{gamma} by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-{gamma} in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-{gamma} in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Typhoid fever, caused by infection with Salmonella enterica serovar typhi, gives rise to considerable morbidity and mortality in developing countries. S. enterica serovar typhimurium (hereafter referred to as S. typhimurium) infection of inbred mice causes a disease that closely resembles human typhoid fever and has been used extensively to model typhoid fever in a laboratory setting (1). Administration of attenuated Salmonella protects susceptible mice from subsequent challenge with virulent Salmonella (2) and therefore mimics immunity conferred by the human typhoid vaccine, Ty21a (3, 4). Greater understanding of the induction of immune responses to Salmonella should therefore aid the development of improved vaccines against human typhoid.

Although Ab responses are known to contribute to Salmonella immunity (5, 6, 7), it is well-established that CD4 T cells and IFN-{gamma} production are critically important for the resolution of infection (8, 9, 10). Clearance of attenuated bacteria occurs several weeks after infection of wild-type (Wt)3 mice, whereas mice with deficiencies in CD4 T cells or IFN-{gamma} production experience uncontrolled bacterial growth (8, 10). Salmonella-specific CD4 T cells are initially activated in the intestine following oral infection (11) or in the spleen after i.v. infection (12). Activated Salmonella-specific CD4 T cells expand, acquire the ability to secrete effector cytokines, and can migrate to infected lymphoid and nonlymphoid tissues to effect bacterial clearance (13, 14, 15). As Salmonella replicates in mononuclear phagocytes, it is typically thought that effector CD4 T cells are activated by recognizing MHC class II (MHC II)/bacterial peptide on the surface of infected cells and then initiate cellular activation via IFN-{gamma} production (16).

However, although CD4 T cell production of IFN-{gamma} is generally considered an adaptive immune response, there are several reports of IFN-{gamma} induction in CD4 T cells by Ag-nonspecific stimuli, particularly involving IL-18 (17, 18, 19, 20). In infection models, it has recently become clear that Ag-experienced effector CD8 T cells acquire the capacity to respond to innate stimuli that can alter, or directly induce, IFN-{gamma} production (21, 22). Such innate activation of T cells may serve an important function in amplifying effector cytokine production at sites of infection, especially if the pathogen is capable of inhibiting Ag presentation in infected cells (23). In mice infected with lymphocytic choriomeningitis virus (LCMV), activation with LPS-induced cytokines was sufficient to induce IFN-{gamma} production from LCMV-specific CD8 T cells in the absence of TCR stimulation (24, 25). These studies suggest a role for IL-12 and IL-18 in this process of innate CD8 activation in infection models (24, 25). Similar results have been documented for CD8 T cell responses to Listeria infection or LPS injection, where memory CD8 T cells were rapidly activated to produce IFN-{gamma}, and have been shown to contribute to protective immunity in the absence of cognate Ag stimulation (26, 27, 28, 29). These reports demonstrate that previously activated CD8 T cells can be induced to produce IFN-{gamma} by cognate Ag or by innate cytokine stimulation.

The possibility that CD4 T cells can secrete effector cytokines in response to innate triggering has not been directly examined, particularly in models of infection where CD4 responses are critical for immunity. We previously described a surprisingly large number of activated CD4 T cells in Salmonella-infected mice that could rapidly produce IFN-{gamma} in vivo (30). We therefore sought to determine whether these CD4 cells could be activated by innate stimuli and whether IL-18 was required for IFN-{gamma} production. In this study, we report that CD4 T cells in Salmonella-infected mice can be activated to secrete IFN-{gamma} via an Ag-independent mechanism that involves IL-18.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mouse strains

IL-18-deficient (B6.129P2-Il18tm1Aki/J), IL-18R-deficient (B6.129P2-Il1rrptm1Aki), and CD90.1 congenic (B6.PL-Thy1a/CyJ) C57BL/6 mice were purchased from The Jackson Laboratory and maintained by intercrossing in our facility. MHC II-deficient mice (B6.129-H2-Ab1tm1GruN12) were purchased from Taconic Farms. C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and used at 6–16 wk of age. All mice were housed in specific pathogen-free conditions and cared for in accordance with University of Minnesota Research Animal Resource guidelines.

Salmonella infection

S. typhimurium AroA–D– (BRD509) were grown overnight in Luria-Bertani broth without shaking and diluted in PBS following estimation of bacterial concentration using a spectrophotometer. Mice were infected i.v. in the lateral tail vein with 5 x 105 bacteria and monitored for signs of infection. In all infection experiments, the actual bacterial dose administered was confirmed by plating serial dilutions onto MacConkey agar plates. To determine bacterial colonization in vivo, spleens from infected mice were homogenized in PBS and serial dilutions plated onto MacConkey agar plates. After overnight incubation at 37°C, bacterial plates were counted and bacterial burdens calculated for each individual organ. Heat-killed S. typhimurium (HKST) was prepared by resuspending bacteria in PBS at a concentration of 5 x 108/ml, heating at 75°C for 1 h, and plating to confirm the absence of live bacteria.

Listeria infection

Listeria and Listeria-OVA (31) were grown overnight at 37°C in tryptic soy broth with shaking. The following day, subcultures were set up, bacterial numbers were estimated using a spectrophotomoter, and mice were infected i.v. with 5 x 103 Listeria. Heat-killed Listeria (HKLM) was prepared by resuspending bacteria at 5 x 108/ml, in PBS and incubating at 75°C for 1 h. Bacteria were routinely plated to confirm the estimated bacterial counts and to confirm the absence of live bacteria in HKLM preparations.

Activation of Ag-specific T cells in vivo

At various times after infection, Salmonella-infected, Listeria-infected, or naive mice were injected i.v. with 1 x 108 HKST, HKLM, or LPS. Spleen cells were harvested 4 h later into Eagle’s Hanks amino acid medium (Invitrogen Life Technologies) containing 2% FBS and 5 mM EDTA and a single-cell suspension generated. Spleen cells were rapidly surface stained at 4°C, before being fixed with formaldehyde, permeabilized using saponin (Sigma-Aldrich), and stained intracellularly using cytokine-specific Abs. Ultra-pure Escherichia coli LPS (Alexis) was used for most experiments except when indicated.

Flow cytometric analysis

Spleen cells were incubated for 20–45 min at 4°C 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 primary Abs. FITC-, PE-, CyChrome-, PE-Cy5-, or allophycocyanin-conjugated Abs specific for CD4, CD11a, CD45.1, CD69, CD90.1, IFN-{gamma}, TNF-{alpha}, and isotype control Abs were purchased from eBioscience and BD Biosciences. OVA-specific CD8 T cells were detected using an H-2Kb tetramer containing the OVA peptide SIINFEKL and was produced as previously described (32). After staining, cells were fixed and analyzed by flow cytometry using a FACSCalibur or a FACS canto. Data were analyzed using FlowJo software (Tree Star).

Adoptive transfer of Salmonella-specific CD4 T cells

CD90.1 congenic C57BL/6 mice were sacrificed 14–30 days after infection with BRD509 and splenic CD4 T cells isolated by negative sorting using magnetic beads (Miltenyi Biotec). CD4 T cells were subsequently injected into C57BL/6 or MHC II-deficient recipients via the lateral tail vein. Before adoptive transfer, the purity of CD4 T cells was determined by flow cytometry after staining with Abs specific for CD4, CD8, B220, and MHC II, and was between 60 and 80% CD4 T cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4 T cells in Salmonella-infected mice can be activated by LPS

A low frequency of CD4 T cells from Salmonella-infected mice produced IFN-{gamma} production directly ex vivo (Fig. 1). However, although few CD4 T cells in naive mice produced IFN-{gamma} in response to HKST, most CD11ahigh CD4 T cells in Salmonella-infected mice were activated to secrete large amounts of IFN-{gamma} in response to HKST injection (Fig. 1, HKST). A proportion of these CD11ahigh CD4 T cells in Salmonella-infected mice also produced TNF-{alpha} in response to HKST (Fig. 1, HKST). This ability of CD4 T cells to secrete IFN-{gamma} and TNF-{alpha} was observed throughout infection and persisted long after bacterial clearance, suggesting that this crude assay allows the detection of an adaptive immune response to Salmonella (30).


Figure 1
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  		FIGURE 1. Innate activation of CD4 T cells in Salmonella-infected mice. C57BL/6 mice were infected i.v. with 5 x 105 Salmonella and 14 days later were injected i.v. with 1 x 108 HKST, 25 µg of LPS, or 25 µg of ultra-pure LPS (UP-LPS). Four hours later, splenocytes were harvested and stained for surface markers and intracellular cytokine production. Naive mice were also injected with HKST or LPS as controls. Each plot shows staining after gating on live CD4 T cells and is representative of three mice per group. This experiment was completed four times with similar results.

 
It was not clear whether injection of HKST activated CD4 T cells in an Ag-specific manner. To examine this issue, we injected naive or Salmonella-infected mice with bacterial LPS, an inflammatory stimulus that is not known to activate CD4 T cells in an Ag-specific manner. Four hours after LPS injection, a large proportion of CD11ahigh, CD4 T cells in Salmonella-infected mice produced IFN-{gamma} while CD4 T cells in naive mice did not (Fig. 1, LPS). These data indicated that CD4 T cells in Salmonella-infected mice had acquired the ability to secrete IFN-{gamma} in response to innate immune activation. An alternative possibility was that LPS preparations were contaminated with bacterial Ags that could be presented in a class II-restricted fashion. However, the injection of ultra-pure LPS activated a similarly large proportion of CD4 T cells in Salmonella-infected mice (Fig. 1, LPS-UP), indicating that such activation could not be attributed to bacterial contaminants. To avoid this possibility, ultra-pure LPS was used for all subsequent experiments. Interestingly, there were two obvious differences between LPS and HKST activation of CD4 T cells in Salmonella-infected mice. First, the frequency of CD4 T cells responding to LPS and the amount of IFN-{gamma} per CD4 cell was consistently lower in response to LPS vs HKST stimulation (Fig. 1), and second, LPS did not cause the production of TNF-{alpha} (Fig. 1). These data suggest that CD4 T cells in Salmonella-infected mice acquire the capacity to produce IFN-{gamma} rapidly in response to innate immune activation, a phenomenon previously reported for CD8 T cells in microbial infection (24, 25, 27).

Innate activation of CD4 T cells is restricted to the period of active infection

Next, we determined the kinetics of innate activation of CD4 T cells in vivo. Initially, we examined spontaneous cytokine production in mice that did not receive ant HKST or LPS injection but were at different stages of Salmonella infection. Significant IFN-{gamma} and TNF-{alpha} production was detected on day 14 but was undetectable at other time points (Fig. 2A). Other groups of C57BL/6 mice were infected with Salmonella and injected with HKST or LPS at various time points to activate CD4 cells. Time points were chosen to reflect important stages in this particular infection model. Day 12 is the peak of bacterial infection, day 40 is the earliest point following sterile clearance, and 3 mo represents a late stage where immune memory is apparent (30).


Figure 2
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  		FIGURE 2. Innate activation of CD4 T cells is most apparent in actively infected mice. C57BL/6 mice were infected i.v. with 5 x 105 Salmonella and on the indicated days after infection were (A) processed for intracellular cytokine staining or (B) injected i.v. with 1 x 108 HKST or 25 µg of ultra-pure LPS (LPS). Day 0 represents uninfected control mice. Four hours later, splenocytes were harvested and stained for surface markers and intracellular cytokine production. Each plot shows staining after gating on live CD4 T cells and is representative of three mice per group. This experiment was completed three times with similar results.

 
At the peak of infection, injection of LPS or HKST stimulated the majority of CD11ahigh CD4 T cells to produce IFN-{gamma}, while only HKST was able to induce TNF-{alpha} production (Fig. 2B, day 12). After bacterial clearance, HKST was able to induce IFN-{gamma} production by approximately two-thirds of CD11ahigh CD4 cells, and TNF-{alpha} production at much lower frequency (Fig. 2B, day 40). At this time point, the response of CD4 T cells to LPS was considerably diminished, with only 28% of this population responding to LPS (Fig. 2B, day 40). At 3 mo following infection, a small population of CD11ahigh cells remained producing IFN-{gamma} and TNF-{alpha} in response to HKST while responsiveness to LPS was completely lost (Fig. 2B, 3 mo). These data indicate that innate activation of CD4 T cells in response to LPS can occur while mice are actively infected with Salmonella, and declines rapidly thereafter.

As noted above, CD4 TNF-{alpha} production is induced by HKST but not by LPS injection at all time points analyzed (Fig. 2B). The proportion of CD4 cells producing TNF-{alpha} is consistently lower than those producing IFN-{gamma}, and only the highest IFN-{gamma} producers actually secrete TNF-{alpha} (data not shown). At 3 mo after infection, a small memory population responds to HKST injection by secreting both IFN-{gamma} and TNF-{alpha} production in the absence of active infection (Fig. 2B). These data suggest that TNF-{alpha} production may be a marker of Ag-dependent CD4 T cell activation while the majority of IFN-{gamma} production may occur by innate activation of CD4 cells in Salmonella-infected mice.

Innate cytokine production induced in Salmonella- and Listeria-infected mice

To examine whether activation of T cells by innate activation occurred in other infections, we examined the effect of HKST injection in Listeria monocytogenes-infected mice. Listeria was chosen because it is a Gram-positive bacterium, making the presence of cross-reactive epitopes with Salmonella unlikely. Mice were infected with Listeria or Salmonella and injected with HKST or HKLM 7 days later. In the absence of any injection, a low level of CD4 IFN-{gamma} and TNF-{alpha} production was observed in Salmonella-infected mice (Fig. 3A), while no IFN-{gamma} or TNF-{alpha} production was detected in CD8 cells from the same mice. At this stage, the frequency of activated CD11ahigh CD4 T cells was considerably lower in Listeria-infected vs Salmonella-infected mice, while the proportion of CD11ahigh CD8 T cells was higher (Fig. 3, no injection). Presumably, this difference reflects the important role of CD8 T cells in clearing Listeria infection and CD4 cells in clearing Salmonella infection (8, 33).


Figure 3
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  		FIGURE 3. HKST induces innate activation of T cells in Listeria-infected mice. C57BL/6 mice were infected i.v. with 5 x 103 Listeria or 5 x 105 Salmonella and 7 days later were injected i.v. with 1 x 108 HKST or 1 x 108 HKLM. Infected mice that were not injected are shown as controls. Four hours later, splenocytes were harvested and stained for surface markers and (A) IFN-{gamma} or (B) TNF-{alpha} production. Each plot shows staining after gating on live CD4 or CD8 T cells and is representative of three mice per group. This experiment was completed three times with similar results.

 
In Salmonella-infected mice, the injection of HKST induced the production of IFN-{gamma} and TNF-{alpha} from CD4, and IFN-{gamma} from CD8 T cells, whereas injection of HKLM did not increase cytokine production above uninjected mice (Fig. 3, HKST). Thus, the inflammatory response to HKLM was insufficient to activate T cells in Salmonella-infected mice. However, injection of HKST induced IFN-{gamma} production, but not TNF-{alpha} production, from CD4 and CD8 T cells in Listeria-infected mice (Fig. 3, HKST). Indeed, HKST served as a better activator of cytokine production in Listeria-infected mice than injection of HKLM (Fig. 3, HKLM). These data demonstrate that HKST can induce innate cytokine production from activated T cells in a completely different infection model. Interestingly, TNF-{alpha} was produced by CD4 T cells in infected mice, only in response to injection of Ag derived from the infecting agent itself. (Fig. 3B, CD4). Again, this suggests that TNF-{alpha} production is not induced in response to innate stimuli and that TNF-{alpha} production may represent Ag-specific activation of effector T cells in vivo.

Innate T cell activation does not require cross-reactive Ag or class II expression

It remained possible that some cross-reactive bacterial Ags were responsible for the HKST activation of CD4 and CD8 T cells in Listeria-infected mice. To rule out this possibility, we visualized the cytokine production of OVA-specific CD8 T cells using class I tetramers. Mice were infected with Listeria-OVA to activate OVA-specific CD8 T cells and these cells detected in the spleen of infected mice. As expected, OVA-specific CD8 T cells had expanded 7 days after infection and displayed an activated phenotype (Fig. 4A). In the absence of HKST injection OVA-specific CD8 cells did not produce IFN-{gamma} (Fig. 4B, no injection). However, the vast majority of these cells produced IFN-{gamma} in response to injection with HKST. These data demonstrate conclusively that HKST injection can activate Listeria-specific CD8 T cells in an Ag-independent manner.


Figure 4
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  		FIGURE 4. Activation of OVA-specific CD8 T cells by HKST. C57BL/6 mice were infected i.v. with 5 x 103 Listeria-OVA and 7 days later were injected i.v. with 1 x 108 HKST. Infected mice that were not injected are shown as controls. Four hours later, splenocytes were harvested and stained for surface markers and (A) an OVA-specific tetramer and (B) an Ab to IFN-{gamma} or isotype control. Plots show staining after gating on live CD8 T cells. Boxed gates show the cytokine production and numbers represent the percentage of CD8 T cells in the gate. This experiment was completed two times with similar results.

 
Next, we examined innate CD4 T cell cytokine production in the absence of MHC II. CD4 T cells were isolated from CD90.1 congenic C57BL/6 mice 14 days after infection with Salmonella, and adoptively transferred into C57BL/6 or MHC II-deficient mice. One day after transfer, mice were injected with LPS or HKST and 6 h later, CD4 cytokine production from transferred cells was examined by gating on CD90.1+ cells. As a positive control, infected CD90.1 mice were also injected with HKST and analyzed for cytokine production. Compared with CD90.1-infected control mice, the frequency of CD11ahigh cells was reduced in all transfer recipients (Fig. 5), perhaps because some of these transferred cells died or migrated to nonlymphoid organs. However, irrespective of the frequency, IFN-{gamma} was produced by CD11ahigh CD4 T cells in response to injection of HKST or LPS in both C57BL/6 and MHC II-deficient recipients (Fig. 5). Therefore, MHC II is not required for innate CD4 T cell activation by HKST. In contrast, although the frequency was very low, TNF-{alpha} appeared to be produced only in response to HKST and was dependent on the expression of MHC II (Fig. 5). Again, these data suggest that TNF-{alpha} production by activated CD4 T cells reflects cognate Ag stimulation rather than innate activation.


Figure 5
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  		FIGURE 5. Innate activation of CD4 T cells occurs in the absence of MHC II. Congenic CD90.1 C57BL/6 mice were infected i.v. with 5 x 105 Salmonella and 21 days later CD4 T cells were purified and transferred to CD90.2 C57BL/6 or CD90.2 MHC II-deficient recipients. One day after transfer, mice were injected with 1 x 108 HKST or 25 µg of ultra-pure LPS. A group of transferred but uninfected mice were used as a negative control (transfer only), and infected, donor CD90.1 mice were injected with 1 x 108 HKST as a positive control (CD90.1 control). Four hours later, splenocytes were harvested and stained for surface markers and intracellular staining for IFN-{gamma}, TNF-{alpha}, or with an isotype control Ab. Plots show staining after gating on live transferred CD90.1+ CD4 T cells. Numbers show the percentage of CD4 T cells within each quadrant. This experiment was completed three times with similar results.

 
Innate CD4 T cell activation in Salmonella-infected mice is dependent upon IL-18

Recent reports suggest that IL-12 and IL-18 can cause innate activation of effector CD8 T cells to produce IFN-{gamma} (21, 22). We therefore examined the contribution of IL-18 to CD4 T cell IFN-{gamma} production in Salmonella-infected mice. IL-18-deficient and IL-18R-deficient mice were infected with Salmonella and the responsiveness to HKST or LPS injection was examined at days 13 and 45 representing the peak of infection and immediately after bacterial clearance. At day 13, a significant proportion of CD11ahigh CD4 T cells from Wt, IL-18-deficient, and IL-18R-deficient mice secreted IFN-{gamma} directly ex vivo, but the frequency of IFN-{gamma} producers was reduced in the absence of IL-18 or IL-18R signaling (Fig. 6A, IFN-{gamma}). A much smaller subset produced TNF-{alpha} spontaneously and the frequency did not differ between Wt and gene-deficient mice (Fig. 6A, TNF-{alpha}). As expected, injection of Wt mice with HKST induced considerable IFN-{gamma} and TNF-{alpha} production from CD11high CD4 T cells (Fig. 6), however, IFN-{gamma} production was reduced considerably in IL-18-deficient and IL-18R-deficient mice (Fig. 6A). Similarly, LPS injection induced IFN-{gamma} production in Wt mice but was much reduced in IL-18-deficient and IL-18R-deficient mice. These data indicate that during active Salmonella infection, IL-18 and IL-18R signaling is required for innate activation of CD4 T cells. However, given the detection of residual LPS-induced IFN-{gamma} production in IL-18 and IL-18R-deficient mice there must also exist IL-18-independent innate activation of CD4 cells. As expected, TNF-{alpha} production was induced by injection of infected mice with HKST, but not with LPS (Fig. 6A). TNF-{alpha} production was also reduced in IL-18 and IL-18R-deficient mice (Fig. 6A), indicating that IL-18 signaling is required for optimal cytokine production in response to HKST.


Figure 6
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  		FIGURE 6. IL-18 contributes to innate activation of CD4 T cells to secrete IFN-{gamma}. Wild-type (WT), IL-18-deficient (IL-18–/–), and IL-18R-deficient (IL-18R–/–) mice were infected i.v. with 5 x 105 Salmonella and (A) 13 or (B) 45 days later after infection, mice were injected with 1 x 108 HKST or 25 µg of ultra-pure LPS. Some infected mice were not injected as a negative control (no injection). Four hours after injection, splenocytes were harvested and stained for surface markers and intracellular cytokine production. Plots show staining after gating on live CD4 T cells in each group of mice and are representative of three mice per group. Numbers show the percentage of CD4 T cells within each quadrant. This experiment was completed three times with similar results.

 
After bacterial clearance (day 45), Wt, IL-18-deficient, and IL-18R-deficient mice displayed no evidence of spontaneous IFN-{gamma} or TNF-{alpha} production ex vivo (Fig. 6B, no injection). However, as observed at the earlier time point, HKST injection induced IFN-{gamma} and TNF-{alpha} production from CD11ahigh CD4 T cells and IFN-{gamma} production was partially dependent on IL-18 and IL-18R expression (Fig. 6B, HKST). Interestingly, LPS-induced IFN-{gamma} production was almost completely dependent on IL-18 and IL-18R expression at this time point (Fig. 6B, LPS). TNF-{alpha} production in response to HKST was unaffected by IL-18 or IL-18R deficiency at this time point. These data suggest that IL-18-mediated innate activation of CD4 T cells occurs maximally at the peak of bacterial infection and gradually decays after bacterial clearance.

IL-18, but not IL-18R, deficiency impairs bacterial clearance

As IL-18 is involved in innate activation of CD4 T cells to produce IFN-{gamma} in Salmonella-infected mice and IFN-{gamma} is required for Salmonella clearance (8), we examined whether bacterial clearance was impaired in IL-18-deficient and IL-18R-deficient mice. Wt, IL-18-deficient, and IL-18R-deficient mice were infected with Salmonella and bacterial burdens in the spleen were determined at several time points after infection. IL-18-deficient mice had consistently higher bacterial counts at every stage of infection while IL-18R-deficient mice had similar bacterial burdens to Wt mice (Fig. 7). These data indicate that IL-18 expression is required for bacterial clearance in vivo.


Figure 7
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  		FIGURE 7. IL-18 contributes to resolution of Salmonella infection. Wild-type (WT), IL-18-deficient (IL-18), and IL-18R-deficient (IL-18R) mice were infected i.v. with 5 x 105 Salmonella and bacterial loads determined in the spleen at 7, 13, and 45 days later. Bars show mean bacterial loads ± SD for six mice per group on day 7, and 12 mice per group on days 13 and 45. This experiment was completed three times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies demonstrated that Ag-experienced CD8 T cells gain the capacity to produce IFN-{gamma} in response to innate stimuli, and that these responses can contribute significantly to pathogen clearance (24, 25, 26, 27, 28, 29). Here, we demonstrate that CD4 T cells develop the capacity for innate activation during Salmonella infection and that this is process is MHC II independent and partially dependent on IL-18.

We initially examined the activation of CD4 T cells in response to HKST injection as a crude assay to quantify endogenous Salmonella-specific T cells in infected mice (30). Quantifying the total endogenous CD4 T cell response to Salmonella has been difficult due to inadequate definition of antigenic targets (34). Our data demonstrate that CD4 T cells in infected mice can be activated in a nonspecific manner by LPS or HKST. However, innate stimuli do not activate a large proportion of memory CD4 T cells in uninfected mice, or naive T cells in Salmonella-infected mice. Furthermore, the number of CD4 cells that are responsive to innate activation is roughly equivalent to the increased frequency of activated CD4 T cells found in the spleen after infection (30). Determining whether these cells are Salmonella-specific or bystander CD4 cells will require the development of novel Ag-specific tools to definitively track endogenous Salmonella-specific T cells in vivo.

Whether these CD4 cells are actually Salmonella-specific or not, our data demonstrate that they are poised to secrete IFN-{gamma} rapidly when exposed to innate stimuli. The ability of ultra-pure LPS to induce IFN-{gamma}, the demonstration that HKST induces IFN-{gamma} in Listeria-infected mice, and the production of IFN-{gamma} by OVA-specific T cells in response to HKST, argue strongly that CD4 activation is not induced by a dominant or cross-reactive Ag presented on MHC II. Although it is possible that exposure to innate stimuli increases the efficiency of endogenous class II processing or expression in Salmonella-infected mice, the ability of LPS and HKST to induce IFN-{gamma} production in MHC II-deficient recipients makes this hypothesis unlikely. Our data are most consistent with the notion that activated CD4 T cells in Salmonella-infected mice acquire the ability to respond to innate stimuli or the products of innate stimuli.

The acquisition of CD4 innate responsiveness was transient and correlated with peak bacterial loads in the spleen. Although IFN-{gamma} production in response to HKST and LPS was detectable immediately following bacterial clearance, it was already declining compared with peak infection, and was absent at a later time point. The capacity of CD8 T cells to respond to innate stimuli during LCMV and Listeria infection suggest that this may be a common process accompanying microbial infection (24, 25, 26, 27, 28, 29). One obvious difference between our study and previous reports is the fact that we have detected CD4 rather than CD8 T cells responding to innate stimuli. This difference may simply reflect the fact that CD4 T cells are expanded more readily during Salmonella infection while CD8 T cells are preferentially amplified during viral or Listeria infection (35, 36). It will be of interest to determine whether CD4 T cells acquire innate responsiveness during Leishmania or mycobacterial infection, models more likely to induce greater CD4 T cell expansion.

The actual components of HKST that contribute to innate activation of CD4 T cells in Salmonella-infected mice are unclear. The fact that LPS administration triggers IFN-{gamma} production suggests that HKST may act via TLR ligation. Indeed, previous reports have determined the effectiveness of LPS and poly(I:C) for innate activation of CD8 T cells (25, 28). The greater efficiency of HKST over LPS may therefore derive from simultaneous activation by multiple TLR ligands, such as flagellin (37), bacterial lipoproteins (38), and CpG DNA (39).

We have demonstrated that innate CD4 activation is at least partly mediated by IL-18 and IL-18R. IL-18 is well-known to induce IFN-{gamma} from CD4 T cells, particularly in combination with IL-12 (17, 19, 40, 41). Previous studies found no alteration in the level of IL-18R expressed by CD8 T cells responding to innate stimuli (25). It seems likely therefore that alteration in T cell responsiveness to IL-18 may be due to synergy with other cytokines such as IL-12 (25). Our studies using IL-18-deficient and IL-18R-deficient mice confirm a role for IL-18 in innate activation of CD4 T cells during Salmonella infection. The CD4 response to LPS was more affected than the response to HKST, perhaps reflecting the fact that a portion of the HKST response involves Ag presentation and TCR ligation. Indeed, TNF-{alpha} production in response to HKST injection follows a pattern more typical of Ag-dependent responses while LPS did not induce TNF-{alpha}. Alternatively, these differences may be explained by the action of alternative TLR agonists in the HKST Ag preparation.

Previous work with IL-18-deficient and caspase-1-deficient (deficient in the production of both IL-18 and IL-1beta) mice have attached differing importance to IL-18 in Salmonella clearance (42, 43, 44). We detected modest increases in bacterial load in IL-18-deficient mice, suggesting that Ag-independent IFN-{gamma} secretion may contribute to bacterial clearance. Interestingly, IL-18R-deficient mice displayed no deficiency in Salmonella clearance mice, which suggests that IL-18 may function via an alternate receptor to IL-18R, or that an alternative ligand for the IL-18R causes increased susceptibility in the absence of IL-18. Indeed, recent work has suggested that an alternative ligand for the IL-18R may be involved in autoimmune inflammatory responses (45, 46).

In conclusion, our data demonstrate that CD4 T cells in Salmonella-infected mice acquire the ability to respond to innate stimuli and that this is partially dependent on IL-18. Understanding whether such innate activation contributes significantly to pathogen clearance may aid the development of novel vaccines and therapeutics against bacterial infection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI056172 (to L.L. and S.J.M.) and AI055743 (to S.J.M.). Back

2 Address correspondence and reprint requests to Dr. Stephen J. McSorley, Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, and Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, McGuire Translational Research Facility, TRF DC 2873, 2001 6th Street SE, Minneapolis, MN 55455. E-mail address: mcsor002{at}umn.edu Back

3 Abbreviations used in this paper: Wt, wild type; LCMV, lymphocytic choriomeningitis virus; MHC II, MHC class II; HKST, heat-killed S. typhimurium; HKLM, heat-killed Listeria. Back

Received for publication December 27, 2006. Accepted for publication March 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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