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Infection and Immunity, April 2009, p. 1492-1501, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01207-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiple Mechanisms Contribute to the Robust Rapid Gamma Interferon Response by CD8+ T Cells during Listeria monocytogenes Infection{triangledown}

Elsa N. Bou Ghanem, Denise S. McElroy, and Sarah E. F. D'Orazio*

Department of Microbiology, Immunology, & Molecular Genetics, University of Kentucky, Lexington, Kentucky

Received 29 September 2008/ Returned for modification 13 December 2008/ Accepted 19 January 2009


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ABSTRACT
 
A subset of CD8+ T cells can rapidly secrete gamma interferon (IFN-{gamma}) in an antigen-independent and interleukin-12 (IL-12)- and IL-18-dependent manner within 16 h of infection with the intracellular bacterial pathogen Listeria monocytogenes. This rapid IFN-{gamma} response is robust enough to be detected directly ex vivo and is not observed following infection with intracellular bacterial pathogens that remain sequestered within host cell vacuoles. We demonstrate here that three distinct pathways can lead to rapid secretion of IFN-{gamma} by CD8+ T cells during L. monocytogenes infection: (i) a direct cytokine-inducing activity encoded by the cholesterol-dependent cytolysin (CDC) listeriolysin O (LLO) acts within the infected cell, (ii) the pore-forming activity of LLO promotes cytosolic localization of bacterial products that trigger cytosol-specific signaling pathways, and (iii) the sustained presence of high concentrations of bacterial products can exogenously trigger cytokine production. Although it has been suggested that CDC protein toxins may act as Toll-like receptor 4 (TLR4) agonists to trigger proinflammatory cytokine secretion, we show in this report that TLR4 signaling is not required to induce a maximal rapid IFN-{gamma} response by CD8+ T cells. The results presented here indicate that multiple mechanisms contribute to the induction of rapid IFN-{gamma} secretion by CD8+ T cells during Listeria infection and that care must be taken when interpreting the results of in vitro assays, since the contribution of each pathway can vary depending on how the assay is performed.


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INTRODUCTION
 
Gamma interferon (IFN-{gamma}) is a multifunctional cytokine that contributes to both innate and adaptive immune responses and is critical for the clearance of intracellular bacterial pathogens such as Listeria monocytogenes. IFN-{gamma} activates macrophages, which leads to enhanced production of antimicrobial oxygen and nitrogen intermediates and limits bacterial spread during infection (33, 41). IFN-{gamma} also increases major histocompatibility complex class I expression on macrophages, which promotes recognition of infected cells by CD8+ cytotoxic T lymphocytes (11). Mice that lack IFN-{gamma} or its receptor are significantly more susceptible to infection with L. monocytogenes (8, 17).

It has long been known that T cells are an important source of IFN-{gamma} during the adaptive phase of the immune response against L. monocytogenes; however, more recently it was demonstrated that a subset of CD8+ T cells can also secrete IFN-{gamma} during the early, innate phase of the immune response. Berg et al. showed that up to 6% of splenic CD8+ T cells were actively secreting IFN-{gamma} 16 h after infection of mice, a robust response that could be detected directly ex vivo without any further stimulation of the T cells (2, 3). All of the IFN-{gamma}+ CD8+ T cells had a "memory" phenotype (CD44hi); however, the rapid IFN-{gamma} response was not antigen specific and instead depended only on the presence of the cytokines interleukin-12 (IL-12) and IL-18 (2).

In addition to CD8+ T cells, many other cell types can rapidly secrete IFN-{gamma} in an IL-12- and IL-18-dependent manner during L. monocytogenes infection, including NK cells, NK-T cells, and NK dendritic cells (2, 39, 44). It is not yet clear whether IFN-{gamma} secretion by any of these splenocyte subpopulations has a particular physiological relevance. However, Berg et al. found that CD8+ T cells were localized within the white pulp of the spleen close to infected macrophages 24 h after L. monocytogenes infection of mice, while NK cells were limited to the red pulp region of the spleen and did not localize with the foci of infection (4). Furthermore, we recently showed that the rapid IFN-{gamma} response by CD44hi CD8+ T cells occurred in some, but not all, strains of mice and that early IFN-{gamma} secretion by CD8+ T cells correlated with a lower bacterial burden in the spleen and liver 3 days postinfection (9). These findings suggest that IFN-{gamma} secreted by CD8+ T cells may be more effective in limiting bacterial growth during the earliest stages of L. monocytogenes infection than IFN-{gamma} secreted by other cell types.

We previously showed that L. monocytogenes cells that were not actively secreting listeriolysin O (LLO) were unable to trigger rapid IFN-{gamma} expression by CD44hi CD8+ T cells during infection of mice (9). LLO is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming protein toxins that are produced by gram-positive bacteria. Each of these toxins is thought to have a similar four-domain structure, and all encode a conserved undecapeptide at the C terminus that is essential for pore formation (23, 43). During intracellular infection, the pore-forming activity of LLO allows L. monocytogenes to escape from phagocytic or endocytic vacuoles into the host cell cytosol, where the bacteria are able to multiply readily (32, 40). Since bacteria that do not express LLO remain trapped in host cell vacuoles and are unable to multiply, we were not able to determine in that study whether LLO itself induced the rapid IFN-{gamma} response or if LLO simply promoted cytosolic localization of the bacteria which indirectly allowed for triggering of a cytosol-specific transcriptional response that led to IFN-{gamma} production (27, 31, 46).

In vitro studies have shown that incubation of murine splenocytes with high concentrations of recombinant LLO (rLLO) or other related toxins results in the rapid accumulation of IFN-{gamma} in the culture supernatant within 24 h as measured in an enzyme-linked immunosorbent assay (ELISA) (19, 35, 36). NK cells were thought to be the primary source of the IFN-{gamma} detected in those assays; however, this was not directly tested and the investigators did not specifically examine CD8+ T cells (36). When macrophages were depleted from the spleen cell cultures, rLLO treatment failed to induce an IFN-{gamma} response (35). This suggested that rLLO triggered splenic macrophages to secrete IL-12 and IL-18, which in turn induced NK cells and/or CD8+ T cells to express IFN-{gamma}. Pretreatment of rLLO with cholesterol to block pore-forming activity did not inhibit the ability of the protein to trigger rapid IFN-{gamma} production (35). It was further shown that either rLLO or the related CDC family member pneumolysin (PLY) could be truncated at the C terminus to remove the hemolytic active site without losing the ability to induce IFN-{gamma} secretion (1, 26). These studies indicated that the N-terminal domains of CDC protein toxins such as LLO encode a cytokine-inducing activity that can directly trigger rapid IFN-{gamma} secretion in vitro in an IL-12- and IL-18-dependent manner.

Exogenous treatment of splenocytes with rLLO or related CDC proteins can also trigger the production of other cytokines, including IL-1{alpha}, IL-6, and tumor necrosis factor alpha (TNF-{alpha}) (20, 35, 36). Park et al. recently showed that treatment of bone marrow-derived macrophages (BMM{phi}) with either LLO, anthrolysin O, perfringolysin O, or seeligeriolysin O resulted in an increase in both IL-6 and TNF-{alpha} mRNA levels and that Toll-like receptor 4 (TLR4) signaling was required to observe this effect (37). In addition, Malley et al. showed that PLY treatment induced peritoneal macrophages to rapidly secrete IL-6 and TNF-{alpha} in a TLR4-dependent manner (30). Based on these observations, it has been suggested that CDC protein toxins may serve as TLR4 agonists in gram-positive bacteria, which lack lipopolysaccharide (LPS), the classically defined receptor for TLR4. IFN-{gamma} expression was not tested in these studies; thus, it is not yet clear whether TLR4 signaling is required to trigger the rapid IFN-{gamma} response we have observed during L. monocytogenes infection.

Although it has been well documented for over a decade that rLLO can induce rapid cytokine production by murine splenocytes in vitro, it is still not clear whether the protein toxin acts outside the host cell, within a host cell vacuole, or in the host cell cytosol. Furthermore, the contributions made by the distinct pore-forming and cytokine-inducing activities of LLO in triggering rapid IFN-{gamma} production during intracellular infection are not well understood. In this study, we present evidence to suggest that at least three different mechanisms of cytokine induction (two dependent on LLO and one LLO independent) can contribute to rapid IFN-{gamma} production by CD8+ T cells during L. monocytogenes infection. We further show that TLR4 signaling is not required to trigger rapid IFN-{gamma} production by CD8+ T cells either in vitro or in vivo.


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MATERIALS AND METHODS
 
Bacteria. Listeria monocytogenes 10403s and Listeria ivanovii 19119 were obtained from the ATCC. The L. monocytogenes hly deletion ({Delta}LLO) strain DP-L2161 (21) was generously provided by Darren Higgins. All Listeria strains were grown in brain heart infusion (BHI) broth (Difco) with shaking at 37°C until early stationary phase, and aliquots were frozen at –80°C. Prior to infection of cells or mice, bacterial aliquots were thawed on ice and grown to early exponential phase with shaking at 37°C in BHI broth. The bacteria were washed once in phosphate-buffered saline (PBS), and dilutions were prepared. Heat-killed (HK) Listeria monocytogenes cells were prepared by incubating exponential-phase L. monocytogenes at 80°C for 2 h. Loss of viable bacteria was verified by plating on BHI agar.

Mice. Five-week-old BALB/c/By/J, C57BL/6/J, C57BL/10SnJ, C57BL/10ScNJ (Tlr4–/–), C3H/HeJ (Tlr4lps-d), and C3H/HeOuJ (Tlr4WT) mice were obtained from The Jackson Laboratory and were maintained in a specific-pathogen-free facility at the University of Kentucky. Mice were used at 6 to 12 weeks of age and all procedures were performed in accordance with the UK IACUC guidelines. For bacterial infections, mice were injected intravenously in the lateral tail vein using a total volume of 200 µl.

Cell culture. For in vitro assays, cells were maintained in antibiotic-free RP-10 medium which consisted of RPMI 1640 (catalog no. 21870; Invitrogen) supplemented with L-glutamine, HEPES, 50 µM 2-mercaptoethanol, and 10% fetal bovine serum (Atlanta Biologicals) and incubated at 37°C in 7% CO2. Single-cell suspensions of splenocytes were prepared by mashing whole spleens through sterile mesh screens using the plunger of a 3-ml syringe. BMM{phi} were generated by plating bone marrow cells harvested from the femurs of mice in BMM-20 medium (Dulbecco's modified Eagle's medium supplemented with L-glutamine, 20% fetal calf serum, 20% L929 supernatant, 50 units/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml gentamicin; catalog no. 11960; Invitrogen) in 24-well dishes (5 x 105 cells/well). The cells were replenished with fresh medium every 4 days and cultured in antibiotic-free medium for 24 h prior to use.

Purification of rLLO protein. The L monocytogenes hly gene (bp 76 to 1590, lacking the signal sequence) was amplified from L. monocytogenes 10403s chromosomal DNA and subcloned into pET15b (Novagen). His-tagged LLO was expressed and purified as described previously (7) using His-Bind resin (Novagen) and stored at 4°C. Purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To block hemolytic activity, protein samples were pretreated with cholesterol at 10 µg/ml for 1 h on ice. Loss of pore-forming activity was verified by testing for hemolysis of sheep red blood cells (RBC) prior to each IFN-{gamma} assay (not shown). Two controls were used to rule out the possibility of LPS contamination triggering IFN-{gamma} production: (i) protein preps were boiled for 10 min prior to use and (ii) some assay wells contained polymyxin B (final concentration, 1 µg/ml).

Hemolysis assay. Serial dilutions of L. monocytogenes culture supernatant filtrates or recombinant proteins were incubated with 10% sheep erythrocytes in an acidic buffer (6 mM cysteine in PBS, pH 5.8) for 60 min at 37°C as described previously (14). Hemolytic activity was defined as the reciprocal of the dilution that was required for 50% hemolysis of the sheep RBC as determined by the absorbance at 514 nm.

ICCS. Intracellular cytokine staining (ICCS) for IFN-{gamma} was performed according to the manufacturer's instructions using the Cytofix/Cytoperm kit (with GolgiPlug; BD Biosciences). For both in vitro and ex vivo assays, the cells were harvested at 14 to 16 h postinfection (hpi) and incubated with the GolgiPlug for 4 h; therefore, cells that were actively secreting IFN-{gamma} either 14 to 18 hpi or 16 to 20 hpi were detected. The following fluorescently conjugated anti-murine antibodies (Abs) were purchased from eBioscience: fluorescein isothiocyanate-conjugated anti-mouse CD8{alpha} (clone 53-6.7), phycoerythrin-conjugated anti-mouse IFN-{gamma} (XMG1.2), and allophycocyanin-conjugated anti-mouse/human CD44 (IM7). Fluorescence intensities were measured using a FACSCalibur flow cytometer, and at least 25,000 events were analyzed using CellQuest software (BD Biosciences). Dead cells and monocytes were excluded on the basis of forward and side scatter.

IFN-{gamma} ELISA. Splenocytes harvested from a naïve C57BL/6 mouse were seeded in a 96-well dish (1 x 106/well) in RP-10 medium lacking antibiotics and then infected with L. monocytogenes at a multiplicity of infection (MOI) of 0.5. Gentamicin was added 90 min later at a final concentration of 25 µg/ml (Gent25) to kill extracellular bacteria. The cell culture supernatants were harvested 24 hpi, and the amount of IFN-{gamma} present was determined by ELISA (eBioscience) according to the manufacturer's instructions.

Filtration of L. monocytogenes culture supernatant. L. monocytogenes (5 x 106 CFU/ml) was incubated in RP-10 medium lacking antibiotics in a 24-well dish at 37°C in 7% CO2 for either 30 or 90 min. Bacteria were pelleted by centrifugation (5 min at 18,000 x g), and the supernatant was collected and passed through a 0.22-µm SpinX filter (Corning). Preliminary studies using both rLLO protein and L. monocytogenes culture supernatants (grown in BHI broth) showed that LLO did not stick to the membrane of the Spin-X filter, based on the results of both a Bradford assay to determine protein concentration and a hemolysis assay to test functional LLO activity (data not shown). Filtrates (500 µl/well) were added to 5 x 106 whole splenocytes per well in RP-10 plus Gent25, and the ICCS was performed as described above.


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RESULTS
 
CD8+ T cells rapidly secrete IFN-{gamma} after incubation with rLLO protein. L. monocytogenes infection triggers a rapid, antigen-independent IFN-{gamma} response by a subset of CD44hi CD8+ T cells that is robust enough to be detected directly ex vivo (2). We recently showed that active secretion of LLO by live L. monocytogenes was required to induce this rapid cytokine response (9). LLO and other members of the CDC family of protein toxins have been shown to have distinct pore-forming and cytokine-inducing activities in vitro, and it is not yet clear which of these activities is required to trigger rapid IFN-{gamma} production during L. monocytogenes infection. Previous studies showed that incubation of murine splenocytes with either truncated or cholesterol-pretreated rLLO proteins that lacked pore-forming activity resulted in accumulation of IFN-{gamma} in the culture supernatant; however, the cell types that secreted IFN-{gamma} in these assays were not clearly defined (35, 36). To test whether rLLO could induce CD44hi CD8+ T cells to rapidly secrete IFN-{gamma}, we incubated murine splenocytes harvested from a naïve C57BL/6 mouse with cholesterol-pretreated rLLO for 14 h and then performed ICCS to determine the percentage of CD8+ T cells that were expressing IFN-{gamma}. As shown in Fig. 1, the number of IFN-{gamma}+ CD8+ T cells increased 11-fold after incubation with rLLO. This result indicates that the cytokine-inducing activity of LLO alone can cause splenic CD8+ T cells to rapidly secrete IFN-{gamma} in the absence of L. monocytogenes infection.


Figure 1
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  		FIG. 1. CD8+ T cells rapidly secrete IFN-{gamma} following exposure to cholesterol-pretreated rLLO protein. Splenocytes harvested from naïve C57BL/6, C3H/HeJ (Tlr4lps-d), or C3H/HeOuJ (Tlr4wt) mice were incubated for 15 h with cholesterol-pretreated rLLO (final concentration, 50 nM) or buffer alone (20 mM Tris), and then ICCS was performed to determine the percentage of IFN-{gamma}+ CD8+ T cells. Dot plots are gated on CD8+ cells, and the numbers in the upper left corners indicate the percentage of CD8+ T cells that were IFN-{gamma}+.

TLR4 signaling is not required to induce rapid IFN-{gamma} secretion by CD8+ T cells. It was recently suggested that CDC proteins such as LLO may be TLR4 agonists and that the cytokine-inducing activity of these protein toxins is mediated through TLR4 signaling (37). To determine whether TLR4 signaling was required for rLLO to induce splenic CD8+ T cells to secrete IFN-{gamma}, we used cells harvested from either C3H/HeJ or C3H/HeOuJ mice. C3H/HeJ mice have a naturally occurring mutation (Tlr4lps-d) that prevents signaling from occurring through TLR4, while C3H/HeOuJ mice have a wild-type TLR4 allele. Splenocytes from each mouse strain were incubated with cholesterol-pretreated rLLO, and the percentage of CD8+ T cells that secreted IFN-{gamma} was determined by ICCS 14 h later. As shown in Fig. 1, CD8+ T cells from both of the C3H mouse strains gave an IFN-{gamma} response that was comparable to CD8+ T cells from C57BL/6 mice. Thus, TLR4 signaling was not required for the cytokine-inducing activity of rLLO to trigger rapid secretion of IFN-{gamma} by CD8+ T cells in vitro.

Since it was possible that exogenous treatment of murine splenocytes with high concentrations of recombinant protein toxin could result in nonphysiologic cytokine induction, we also infected naïve C3H/HeJ or C3H/HeOuJ splenocytes with L. monocytogenes and determined the amount of IFN-{gamma} that had accumulated in the culture supernatant 24 h later by ELISA. As shown in Fig. 2A, no significant difference was observed for cells isolated from either the TLR4 signaling-deficient mice or mice with a wild-type TLR4 allele. Although C3H/HeJ and C3H/HeOuJ mice are closely related strains of mice that are commonly used to compare the effects of TLR4 signaling, they are not genetically identical. Therefore, to further confirm our findings, we also tested CD8+ T cells isolated from TLR4 knockout mice and the parental control strain, C57BL/10. Again, CD8+ T cells harvested from both the wild-type and the TLR4–/– mice showed a significant increase in IFN-{gamma} expression following in vitro infection with L. monocytogenes (Fig. 2B).


Figure 2
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  		FIG. 2. TLR4 signaling is not required to induce a rapid IFN-{gamma} response by CD8+ T cells. (A) Splenocytes from the indicated mouse strains were infected with L. monocytogens (MOI, 0.5) for 90 min, and then gentamicin was added at a final concentration of 25 µg/ml to kill extracellular bacteria. Culture supernatants were collected 24 hpi and the amount of IFN-{gamma} present was determined by ELISA. Mean values ± standard deviations for triplicate samples are shown. (B) Splenocytes harvested from naïve mice were infected with L. monocytogenes as described above, and IFN-{gamma} ICCS was performed 14 h later. The percentage of CD8+ T cells that were IFN-{gamma}+ is shown. (C) Mice were infected with 1 x 106 CFU of L. monocytogenes (15 hpi) or given PBS (naïve). Spleens were harvested 15 h later and the percentage of IFN-{gamma}+ CD8+ T cells was determined directly ex vivo by ICCS. Mean values ± standard deviations for groups of three mice each are shown. (D) Mice (n = 3) were injected with either PBS (naïve) or 5 µg of E. coli LPS (Sigma). Spleens were harvested 4.5 h later and IFN-{gamma} ICCS was performed. For each panel, data from one of two independent experiments are shown.

To verify that TLR4 signaling was not required to induce rapid IFN-{gamma} expression by CD8+ T cells during in vivo infection of mice, we harvested splenocytes 15 h postinfection and tested directly ex vivo for the presence of IFN-{gamma}+ T cells. For these experiments, groups of C3H/HeJ, C3H/HeOuJ, C57BL/10 (B10), and TLR4–/– mice were infected intravenously with 106 CFU of L. monocytogenes and uninfected mice were given PBS alone. BALB/c and C57BL/6 mice were included as controls, since we had previously identified these mice as either IFN-{gamma} "nonresponders" or "high responders," respectively (9). All of the C3H and B10 mouse strains showed a significant increase in the number of IFN-{gamma}+ CD8+ T cells 15 h postinfection, and there was no significant difference in the percentage of IFN-{gamma}+ CD8+ T cells detected in the spleens of mice expressing either wild-type TLR4 or mutant TLR4 alleles (Fig. 2C). As a control, we also injected groups of B10 mice with LPS, since it was previously shown that intravenous administration of LPS could trigger CD8+ T cells in C57BL/6 mice to secrete IFN-{gamma} within 4 to 6 h (22). As shown in Fig. 2D, CD8+ T cells from wild-type mice rapidly secreted IFN-{gamma} within 5 h of LPS injection, while the Tlr4–/– mice showed no IFN-{gamma} response. Together, these results suggest that the direct cytokine-inducing activity of LLO can trigger rapid IFN-{gamma} secretion by CD8+ T cells and that TLR4 signaling is not required for cytokine induction to occur through this pathway or during L. monocytogenes infection.

L. ivanovii infection triggers a weak but detectable rapid IFN-{gamma} response by CD8+ T cells. The results presented above indicate that LLO can trigger IFN-{gamma} secretion by CD8+ T cells even when the pore-forming activity of the toxin is inhibited. (26). However, during infection, the hemolytic activity of LLO promotes phagosomal escape of L. monocytogenes, and it has been shown that cytosolic localization of bacteria induces a unique transcriptional profile that could also lead to cytokine induction (27, 31, 46). Thus, in vivo, it is possible that the pore-forming activity of LLO alone could indirectly trigger the rapid IFN-{gamma} response by promoting the cytosolic localization of bacterial products derived from L. monocytogenes (for example, CpG DNA). To test whether cytosolic localization of bacteria alone was capable of inducing the rapid IFN-{gamma} response, we infected mice with Listeria ivanovii. L. ivanovii is a veterinary pathogen that encodes homologs of all the major virulence proteins found in L. monocytogenes, including ivanolysin O (ILO), a cholesterol-dependent cytolysin that is closely related to LLO (6). ILO contains the conserved C-terminal undecapeptide that codes for pore-forming activity and thus allows for phagosomal escape and cytosolic localization of L. ivanovii (10). However, in vitro studies have shown that purified rILO does not directly induce IFN-{gamma} secretion by murine splenocytes (25). Therefore, during L. ivanovii infection, bacteria can multiply in the cytosol and allow for induction of cytosolic signaling pathways, but there would be no direct cytokine-inducing activity expressed by ILO.

Splenocytes were harvested from C57BL/6 mice 15 h after infection with either L. monocytogenes or L. ivanovii and the number of IFN-{gamma}+ CD8+ T cells was determined directly ex vivo by ICCS. As a control, we also infected mice with a LLO deletion mutant strain of L. monocytogenes ({Delta}LLO). These bacteria lack both the direct cytokine-inducing and the cytosolic localization activities associated with LLO and remain trapped within phagocytic vacuoles (12, 23). As shown in Fig. 3, CD8+ T cells isolated from mice infected with 106 CFU of L. ivanovii did not display a rapid IFN-{gamma} response. However, since it was previously reported that L. ivanovii is approximately 100-fold less virulent in mice than L. monocytogenes (42), we also infected a group of mice with 108 CFU of L. ivanovii to achieve a comparable level of infection as L. monocytogenes. The higher dose of L. ivanovii triggered a weak but detectable IFN-{gamma} response that was significantly greater than the response observed for either naïve mice or mice infected with 108 CFU of {Delta}LLO L. monocytogenes. These results indicate that cytosolic localization of Listeria species alone is sufficient to induce a minimal rapid IFN-{gamma} response by CD8+ T cells and further suggest that the distinct pore-forming and cytokine-inducing activities encoded within LLO can each trigger a pathway that leads to rapid IFN-{gamma} secretion during L. monocytogenes infection.


Figure 3
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  		FIG. 3. L. ivanovii infection triggers a minimal rapid IFN-{gamma} response by CD8+ T cells. Groups of C57BL/6 mice (n = 3) were infected intravenously with either L. monocytogenes (Lm), L. ivanovii (Li), or an hly deletion mutant strain of L. monocytogenes ({Delta}LLO) at the indicated doses. Naïve control mice received injections of PBS alone. Spleens were harvested 15 hpi and the percentage of CD8+ T cells secreting IFN-{gamma} was determined by ICCS. Average values ± standard deviations from one of three separate experiments are shown. *, mean significantly different from either naïve mice or mice infected with {Delta}LLO L. monocytogenes (P ≤ 0.002).

The sustained presence of bacterial products derived from killed L. monocytogenes can exogenously trigger rapid IFN-{gamma} secretion by CD8+ T cells. To further characterize the exact mechanisms involved in triggering the two distinct pathways of LLO-mediated IFN-{gamma} secretion, we needed to develop an in vitro assay that would closely mimic the IFN-{gamma} responses we had observed directly ex vivo. For the experiments shown in Fig. 1, we had simply incubated nonadherent, whole splenocytes with L. monocytogenes for 90 min and then added gentamicin to the medium to kill extracellular bacteria, and it is likely that a variety of cell types were infected simultaneously. But during intravenous infection of mice, bacteria enter the spleen via the red pulp, which contains a large number of phagocytes that serve to remove particulate matter from the circulation. Therefore, splenic macrophages or dendritic cells are likely to be the first cell type infected with L. monocytogenes prior to interaction with lymphocytes, such as CD8+ T cells, in the white pulp of the spleen (34). To develop an in vitro assay that more closely mimics this timeline, we infected BMM{phi} with L. monocytogenes for 30 min, washed the cells, and then added splenocytes from a naïve C57BL/6 mouse as a source of CD8+ T cells. The cells were harvested 14 h postinfection and the number of IFN-{gamma}+ CD8+ T cells was determined by ICCS. Interestingly, the magnitude of the IFN-{gamma} response was significantly lower when we infected the macrophages first and then added naïve splenocytes (Fig. 4B) compared to infection of whole splenocytes (Fig. 4A).


Figure 4
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  		FIG. 4. Heat-killed L. monocytogenes alone can exogenously trigger rapid IFN-{gamma} production in vitro. BMM{phi} monolayers (B, D, and F) or nonadherent splenocytes (A, C, and E) were infected with wild-type L. monocytogenes at an MOI of 0.5 in 24-well dishes. Uninfected cells were incubated in medium alone. BMM{phi} were washed by aspirating medium from the culture well three times and replacing with prewarmed PBS. Splenocytes were transferred to a conical tube and washed three times with prewarmed PBS and then replated in assay wells that had also been washed three times with PBS. Where indicated, gentamicin was added at a final concentration of 25 µg/ml (Gent25). IFN-{gamma} ICCS was performed 15 hpi. Dot plots are gated on CD8+ cells and the percentage of IFN-{gamma}+ cells in the smaller boxes is indicated in the upper left corner of each plot. Dot plots shown are representative of at least three separate experiments performed.

We reasoned that the increased response shown in Fig. 4A could be due to the sustained presence of bacterial products in the assay well, since the infected whole splenocytes (Fig. 4B) were not washed during the assay. Single-cell suspensions of splenocytes consist primarily of lymphocytes, and we previously showed that the efficiency of infection for these nonadherent cells is much less than for infection of adherent BMM{phi} (D.S. McElroy, T. J. Ashley, and S. E. F. D'Orazio, unpublished observations). Therefore, even at a low MOI of 0.5, we would expect there to still be a large number of extracellular bacteria present 90 min postinfection of whole splenocytes. The addition of gentamicin at 25 µg/ml would kill all of these extracellular organisms, but without further washes, it was likely that a high concentration of bacterial debris remained in the assay well. The bacterial debris would contain many ligands that could be recognized by TLR on the surface of dendritic cells, and this could lead to cytokine secretion in the absence of live bacteria. To limit the amount of extracellular bacteria present in the assay mixture, we repeated the experiments but removed the infected splenocytes from the assay well and washed the cells three times in a conical tube. The washed cells were resuspended in fresh medium containing gentamicin and returned to the corresponding assay wells that had also been washed three times with PBS (Fig. 4C). Since washing adherent BMM{phi} in the assay well was not as efficient as washing cells in a conical tube (data not shown), we washed the cells three times, added medium containing gentamicin, and then washed the cells again 30 min later to remove bacterial debris before naive splenocytes were added (Fig. 4D). In both cases, reducing the amount of extracellular bacteria present in the assay well resulted in lower IFN-{gamma} responses and the number of IFN-{gamma}+ CD8+ T cells was similar whether we infected macrophages or whole splenocytes (Fig. 4C and D). These results suggested that the presence of extracellular bacteria or bacterial products derived from killed bacteria could magnify the basal IFN-{gamma} response that was triggered by live infection of cells.

To determine whether killed bacteria or bacterial debris alone could trigger the rapid IFN-{gamma} response by CD8+ T cells in the absence of live L. monocytogenes, we incubated either macrophages or whole splenocytes with HK L. monocytogenes and then determined the number of IFN-{gamma}+ CD8+ T cells 14 h later by ICCS. As shown in Fig. 4E, persistent exposure of whole splenocytes to killed bacteria alone did trigger a significant IFN-{gamma} response. However, adding HK L. monocytogenes to macrophages for 30 min prior to the addition of spleen cells did not induce IFN-{gamma} production by CD8+ T cells (Fig. 4F). These results indicated that prolonged exposure of splenocytes to a high concentration of L. monocytogenes bacterial debris can trigger a rapid IFN-{gamma} response from CD8+ T cells even in the absence of live L. monocytogenes.

Extracellular L. monocytogenes cells do not secrete enough LLO to induce the rapid IFN-{gamma} response. Since we showed, as described above, that high concentrations of rLLO alone could trigger rapid IFN-{gamma} secretion by CD8+ T cells in the absence of live bacteria, it was possible that extracellular LLO secreted by the bacterial inoculum used to infect the cells in our in vitro assays was directly responsible for the IFN-{gamma} responses we observed. LLO expression is tightly regulated in L. monocytogenes and increases significantly during intracellular infection (43); however, low levels of LLO can be found in the supernatant of L. monocytogenes broth cultures grown at 37°C. To determine if there was a sufficient amount of LLO secreted into the culture medium to trigger rapid IFN-{gamma} production, we added the same number of bacteria used in the assays described above to assay wells without cells. The bacteria were incubated in tissue culture medium lacking antibiotics at 37°C in 7% CO2 for either 30 or 90 minutes, the same time periods that were used to infect BMM{phi} and whole splenocytes, respectively. The medium was then passed through a 0.22-µm filter to remove the bacteria and the filtrate was then added directly to naïve splenocytes. As shown in Fig. 5, neither filtrate was capable of inducing CD8+ T cells to rapidly secrete IFN-{gamma}. We also tested the filtrates for the ability to lyse sheep red blood cells in an acidic buffer, but we were unable to detect any hemolytic activity (data not shown). These data indicate that the bacterial inocula used in our in vitro assays did not secrete enough functionally active LLO to induce rapid cytokine secretion in the absence of infection and are consistent with results of previous studies that showed that only high concentrations (50 to 100 nM) of rLLO protein trigger cytokine production in vitro (35, 36).


Figure 5
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  		FIG. 5. Exponential-phase L. monocytogenes does not secrete enough LLO into tissue culture medium to exogenously trigger a rapid IFN-{gamma} response. L. monocytogenes (5 x 106 CFU/ml) was incubated in RP-10 lacking antibiotics in a 24-well dish at 37°C in 7% CO2. Either 30 or 90 min later the medium was harvested and either used directly or passed through a 0.22-µm filter to remove the bacteria (filtrate). RP-10 medium alone, L. monocytogenes-containing medium, or filtrate was then added directly to whole splenocytes from a naïve C57BL/6 mouse. The percentage of CD8+ T cells secreting IFN-{gamma} was determined 15 h later by ICCS and is indicated by the number in the upper left corner of each plot. Dot plots (gated on CD8+ cells) from one of two separate experiments are shown.

In vitro infection of macrophages, but not whole splenocytes, results in rapid IFN-{gamma} secretion that mimics the ex vivo responses observed following infection of mice. The results described above indicated that our in vitro assays had the potential to detect IFN-{gamma} expression triggered by three distinct pathways: (i) direct cytokine induction by LLO, (ii) cytosolic localization of L. monocytogenes indirectly mediated by LLO, and (iii) the sustained presence of exogenous bacterial products. To further define the physiological relevance of infecting BMM{phi} and then adding splenocytes versus infection of whole splenocytes, we used either L. ivanovii or {Delta}LLO L. monocytogenes to infect the cells. Infection of whole splenocytes (as described in Fig. 4B) resulted in a significant number of IFN-{gamma}+ CD8+ T cells following exposure to all three of the bacterial strains (Fig. 6). Infection with either L. monocytogenes or L. ivanovii gave the highest responses, with approximately 4.5 to 5.5% of the CD8+ T cells rapidly secreting IFN-{gamma}. Infection with {Delta}LLO L. monocytogenes gave a slightly lower but still robust response, with 2.6% of the CD8+ T cells staining positively for IFN-{gamma}. Since {Delta}LLO L. monocytogenes cells do not actively secrete a CDC protein such as ILO or LLO, all of this response can be attributed to the presence of bacterial debris in the assay well. These results were not consistent with the percentages of IFN-{gamma}+ cells we observed directly ex vivo when L. ivanovii infection triggered only a weak response and {Delta}LLO L. monocytogenes infection did not induce any significant IFN-{gamma} response (Fig. 3). In contrast, infection of BMM{phi} in vitro followed by the addition of naïve splenocytes gave results that were strikingly similar to the ex vivo responses we observed. As shown in Fig. 6, infection with L. ivanovii triggered a weaker IFN-{gamma} response than L. monocytogenes, and infection with {Delta}LLO L. monocytogenes did not result in a detectable number of IFN-{gamma}+ cells. Thus, the infected macrophage assay more closely mimics the responses observed during infection of mice.


Figure 6
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  		FIG. 6. In vitro infection of BMM{phi} and subsequent addition of CD8+ T cells triggers IFN-{gamma} secretion that closely mimics the IFN-{gamma} responses observed during infection of mice. Splenocytes (5 x 106/ml) harvested from a naïve C57BL/6 mouse were infected with either L. monocytogenes, L. ivanovii, or {Delta}LLO L. monocytognes (MOI, 0.5) as indicated in Fig. 4A. C57BL/6 BMM{phi} (5 x 105/well) were infected as indicated in Fig. 4D. IFN-{gamma} ICCS was performed 15 hpi. The dot plots shown are gated on CD8+ cells; numbers in the upper left corner indicate the percentage of IFN-{gamma}+ CD8+ T cells. Representative data from one of five different experiments are shown.


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DISCUSSION
 
CD8+ T cells secrete IFN-{gamma} during two distinct phases of the primary immune response against L. monocytogenes. A subset of partially activated (CD44hi) T cells begins to produce IFN-{gamma} within 16 h of infection in an antigen-independent process that requires the presence of both IL-12 and IL-18. Specific recognition of peptide antigens also leads to IFN-{gamma} secretion by CD8+ T cells; however, this wave of cytokine production peaks at 5 to 8 days postinfection and is not dependent on the presence of IL-12 and IL-18. In this report, we demonstrate that at least three different pathways can lead to rapid antigen-independent IFN-{gamma} secretion by CD8+ T cells during L. monocytogenes infection: (i) a direct cytokine-inducing activity encoded by LLO operates within infected cells, (ii) the pore-forming activity of LLO indirectly induces cytokine production by allowing for cytosolic localization of bacterial products, and (iii) the sustained presence of high concentrations of degraded L. monocytogenes can exogenously trigger IFN-{gamma} secretion by CD8+ T cells. Our results are consistent with the model shown in Fig. 7, which predicts that each of the distinct mechanisms of cytokine induction is triggered during intracellular infection with L. monocytogenes and that as a result, there is an additive or synergistic effect that leads to the uniquely robust IFN-{gamma} response that has been detected directly ex vivo following infection of mice.


Figure 7
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  		FIG. 7. Proposed model to explain why L. monocytogenes infection induces a robust rapid IFN-{gamma} response. Three distinct pathways can lead to rapid, IL-12- and IL-18-dependent secretion of IFN-{gamma} by CD8+ T cells. (1) LLO expression is upregulated in the host cell phagocytic vacuole. An N-terminal domain of the LLO directly induces IL-12 and IL-18 secretion, possibly by promoting the clustering of host cell signaling molecules present on the vacuolar membrane (13). (2) The pore-forming activity of LLO promotes bacterial escape from the phagosome, and L. monocytogenes cells multiply in the cytosol. Bacterial products such as CpG DNA (or possibly LLO itself) trigger cytosolic signaling pathways that lead to IL-12 and IL-18 production. These pathways could involve multiple cytosolic receptors, including NOD2, Ipaf, or NALP3 (27, 46). (3) Bacterial growth in the cytosol eventually leads to lysis of infected M{Phi}, and high concentrations of the released bacteria or bacterial products may be present in the local microenvironment. Dendritic cells (DC) that have migrated to the site of infection express high levels of TLR proteins and other pattern recognition molecules and can become activated following direct contact with L. monocytogenes cells and L. monocytogenes-derived products. The activated dendritic cells produce additional IL-12 and IL-18 which serve to amplify the rapid IFN-{gamma} response. Only partially activated (CD44hi) CD8+ T cells with a "memory" phenotype can rapidly respond to the presence of IL-12 and IL-18 in an antigen (Ag)-independent manner.

The direct cytokine-inducing effects of LLO and most other CDC proteins except for ILO have been demonstrated primarily by incubating recombinant protein with whole splenocytes in vitro and measuring the amount of IFN-{gamma} that accumulates in the supernatant by ELISA without identifying the cell type(s) that served as the source of the IFN-{gamma}. We showed in this paper that rLLO could induce CD8+ T cells to rapidly secrete IFN-{gamma}, providing a formal link between numerous in vitro studies using rLLO (26, 35, 36) and the more recent data from our lab and James Forman's group showing that IFN-{gamma}+ CD8+ T cells can be detected directly ex vivo within 16 to 18 h of L. monocytogenes infection (2, 3, 9). The physiological significance of in vitro assays using recombinant protein is still not clear, since IFN-{gamma} production appears to be triggered only when rLLO is added at a narrow range of relatively high protein concentrations (25 to 50 nM, or ~3 µg/ml) (19, 26). Furthermore, since the rLLO was added exogenously to splenocytes in all of these assays, it is not clear whether the toxin acted primarily outside the cell or in a subcellular compartment. We demonstrated in this report that extracellular L. monocytogenes did not secrete enough LLO into culture medium to trigger a rapid IFN-{gamma} response in vitro, which suggests that LLO must be present either in the host cell vacuole or cytosol to trigger cytokine production.

In addition to inducing IFN-{gamma} secretion, LLO has been shown to have a variety of other effects on mammalian cells. For example, in vitro stimulation with rLLO caused an increase in mucin formation and IL-6 production by intestinal epithelial cells (29, 44), induced activation of NF-{kappa}B signaling in endothelial cells (24, 37), triggered IL-6 and TNF-{alpha} production from BMM{phi} (37), and induced apoptosis in activated lymphocytes (5). Many of these same effects have been noted following treatment with other CDC protein toxins, such as PLY or anthrolysin. It was recently demonstrated that production of IL-6 or TNF-{alpha} following exogenous treatment with recombinant CDC proteins required TLR4 signaling and, thus, it was suggested that LLO and other members of the CDC family of proteins may be TLR4 agonists (37). We showed here that TLR4 signaling was not necessary either to mediate the direct cytokine-inducing activity of rLLO or to achieve a maximal rapid IFN-{gamma} response following infection with L. monocytogenes. Our studies do not rule out the possibility that TLR4 signaling may be important to induce the expression of other proinflammatory cytokines, such as IL-1, IL-6, or TNF-{alpha}, but do indicate that TLR4 is not an essential component of the signaling cascade that leads to rapid IL-12 and IL-18 production during L. monocytogenes infection.

The pore-forming activities of LLO and other CDC proteins promote bacterial escape from host cell vacuoles and thus indirectly allow for triggering of cytosolic signaling pathways during intracellular infection. We showed here that infection with ILO-expressing L. ivanovii induced a rapid IFN-{gamma} response that was significantly lower than that observed following L. monocytogenes infection, yet it was still strong enough to be detected directly ex vivo. These data are consistent with the recently reported in vitro studies by Hara et al. who used isogenic L. monocytogenes strains expressing either ILO or LLO (15, 16). They showed that the ILO-expressing L. monocytogenes strain triggered a significantly weaker IFN-{gamma} response than the LLO-expressing strain and concluded from these studies that the pore-forming activities encoded within both LLO and ILO were not important for cytokine induction. Our interpretation of these collective observations is that cytosolic localization of bacteria alone can trigger rapid IL-12- and IL-18-dependent IFN-{gamma} secretion and that the intracellular presence of an additional cytokine-inducing activity encoded within LLO can greatly enhance the overall IFN-{gamma} response. One role for the direct cytokine-inducing activity of LLO may be to increase the amount of IL-18 produced during L. monocytogenes infection, as recently suggested by Hara et al. (16). It is interesting that Burkholderia pseudomallei infection can also trigger a rapid IFN-{gamma} response by CD8+ T cells (28). B. pseudomallei is a gram-negative intracellular bacterial pathogen that appears to use type III secreted effector proteins to escape from phagocytic vacuoles into the host cell cytosol in a manner similar to Shigella flexneri (38), and it does not encode a protein with homology to the CDC family of protein toxins.

The physiological relevance of the third pathway leading to rapid IFN-{gamma} secretion by CD8+ T cells has yet to be established. Although heat-killed L. monocytogenes induced a significant IFN-{gamma} response in some of our in vitro assays, we were not able to detect an increase in IFN-{gamma}+ CD8+ T cells directly ex vivo following injection of mice with heat-killed bacteria (9). The in vitro data suggest that prolonged exposure to high concentrations of bacterial products may be needed to induce cytokine production by this pathway. However, it may be that a particular subpopulation of splenocytes other than macrophages needs to be exposed to bacterial products to trigger this mechanism. We postulate that bacterial products found in the heat-killed inoculum interacted with TLR proteins expressed on the surface of dendritic cells to initiate a signaling cascade that led to the production of IL-12 and IL-18 (Fig. 7). Alternatively, the killed bacteria could be degraded in the phagocytic vacuoles of activated macrophages in a manner that leads to triggering of cytosolic signaling cascades as recently described by Hersokovits et al. (18). Regardless of the mechanism involved, it is not yet clear whether there is a local microenvironment in vivo that would replicate the conditions achieved within the culture wells of our "infected splenocytes" in vitro assay. Therefore, care must be taken when interpreting the results of in vitro assays, since we demonstrated here that the exogenous HK L. monocytogenes pathway of cytokine induction can account for a significant number of the total IFN-{gamma}+ CD8+ T cells, depending on how the assay is performed.

We previously showed that mice infected intravenously with Streptococcus pneumoniae did not trigger a rapid IFN-{gamma} response that could be detected directly ex vivo (9). S. pneumoniae expresses the CDC protein PLY, and several cell wall components of the bacteria could be recognized by TLR on the surface of dendritic cells; however, S. pneumoniae is not an intracellular pathogen. These observations suggest that intracellular localization of CDC protein toxins and/or other bacterial products is critical for rapid IFN-{gamma} induction and that recognition of exogenous bacterial products by TLR proteins alone is not sufficient to trigger a robust IFN-{gamma} response in vivo. Kambayashi et al. saw a significant number of IFN-{gamma}+ CD8+ T cells in C57BL/6 mice 3 h after injection of 50 µg of LPS (22). However, intravenous infection of mice with high doses of LPS-containing bacteria such as Escherichia coli or Salmonella enterica serovar Typhimurium did not trigger a rapid IFN-{gamma} response in C57BL/6 mice (9). Thus, the bioavailability of the stimulus (either CDC protein or TLR ligand) during infection appears to be critical for producing enough IL-12 and IL-18 to trigger CD8+ T cells to rapidly secrete IFN-{gamma}. Presumably, the unique intracellular life cycle of L. monocytogenes results in optimal presentation of the appropriate bacterial products in the necessary subcellular locations to maximally induce each of the three distinct pathways leading to rapid IFN-{gamma} production in the spleen. Further studies will be needed to clarify whether there is an additive or synergistic effect that results in the uniquely robust rapid IFN-{gamma} response observed during L. monocytogenes infection of mice.


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ACKNOWLEDGMENTS
 
We thank Greg Baumann for technical assistance and Beth Garvy for critical review of the manuscript.

This work was supported by a grant from the National Center for Research Resources (P20 RR20171) to S.E.F.D.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Immunology, & Molecular Genetics, University of Kentucky, 800 Rose Street, MS415, Lexington, KY 40536. Phone: (859) 323-8701. Fax: (859) 257-8994. E-mail: sarah.dorazio{at}uky.edu Back

{triangledown} Published ahead of print on 29 January 2009. Back

Editor: J. L. Flynn


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Infection and Immunity, April 2009, p. 1492-1501, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01207-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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