Protective T cell response against intracellular pathogens in the absence of Toll-like receptor signaling via myeloid differentiation factor 88

Mischo Kursar1, Hans-Willi Mittrücker1, Markus Koch1, Anne Köhler1, Marion Herma1 and Stefan H. E. Kaufmann1

1 Department of Immunology, Max-Planck-Institute for Infection Biology, Schumannstrasse 21/22, 10117 Berlin, Germany

The first two authors contributed equally to this work
Correspondence to: H.-W. Mittrücker; E-mail: mittruecker{at}mpiib-berlin.mpg.de
Transmitting editor: S. Akira


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLR) have been indicated as germline-encoded receptors for sensing a variety of pathogens. Although the role of TLR in innate immunity is beyond question, their function in acquired immunity, in particular in T cell immunity, is less clear. Here, we used experimental Listeria monocytogenes infection of mice to analyze requirements for TLR2, TLR4 and the central TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the generation of specific T cell responses. We demonstrate that following L. monocytogenes infection, mice deficient in TLR2, TLR4 and MyD88 can generate Listeria-specific CD8+ and CD4+ Th1 responses. These T cell responses are sufficient to control secondary infection with a high dose of L. monocytogenes even in the absence of TLR signaling via MyD88. Thus, TLR2-, TLR4- and MyD88-dependent signals are not essential for the generation of CD4+ Th1 and CD8+ T cells, and T cells can protect mice against infection in the absence of these signals.

Keywords: bacterial infection, Listeria monocytogenes, T cell, Toll-like receptor activation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mammalian immune system has evolved germline-encoded receptors for pathogen-associated molecular patterns (PAMP). Using PAMP receptors, cells of the immune system can sense conserved structures derived from viral, bacterial and eukaryotic pathogens. Recognition of these structures not only causes activation and differentiation of cells directly involved in innate immunity, but also maturation of professional antigen-presenting cells, which eventually initiate adaptive immunity (1,2). Toll-like receptors (TLR) represent a family of related PAMP receptors. Members of the TLR family recognize a variety of conserved pathogen structures such as lipopolysaccharide (TLR4), lipoproteins, peptidoglycans and lipoarabinomannans (TLR2), flagellin (TLR5) or unmethylated CpG motifs (TLR9) (1,3). Triggering of TLR activates different kinase pathways and transcription factors of the NF{kappa}B family. A crucial element of these signaling pathways is the myeloid differentiation factor 88 (MyD88). The central adaptor protein MyD88 converts the triggering of TLR and members of the IL-1 receptor family into intracellular signals, which activate the kinase and NF{kappa}B pathways. More recently, it has been demonstrated that TLR3 and TLR4 can activate NF{kappa}B and the expression of type I IFN via a MyD88-independent pathway. The composition of the MyD88-independent pathway is not clear yet, but most likely involves the adaptor protein TRIF/TICAM-1 and non-canonical I{kappa}B kinases (1,4).

In macrophages and dendritic cells, TLR-mediated signals induce activation and differentiation, leading to the secretion of a variety of cytokines, including IL-12, tumor necrosis factor (TNF)-{alpha} and various chemokines. Macrophages acquire anti-microbial mechanisms such as the production of reactive nitrogen and oxygen radicals. In dendritic cells, TLR stimulation induces maturation into fully competent antigen-presenting cells, accompanied by enhanced expression of adhesion molecules, MHC class II and co-stimulatory ligands. Thus TLR signals are involved in the innate immune response to pathogens, as well as in the initial steps of the acquired immune response, the priming of naive T cells (1,3).

Mice deficient in TLR2, TLR4 and MyD88 have been used to analyze the function of TLR in infections with different pathogens [reviewed in (3)]. In particular, MyD88-deficient mice demonstrate increased susceptibility to a variety of bacterial and eukaryotic pathogens such as Listeria monocytogenes, Staphylococcus aureus, Leishmania major and Toxoplasma gondii (59). In most of these experimental infection models, increased susceptibility of mice was due to a failure in eliciting a protective anti-microbial innate immune response (59). Analysis of T cell responses in MyD88-deficient mice revealed impaired Th cell differentiation. Immunization of mice with helminth antigen or with ovalbumin (OVA) in alum elicited a normal Th2 cell response with CD4+ T cells producing IL-4, IL-5 and IL-13 after antigen re-stimulation (10,11). In contrast, immunization studies in MyD88–/– mice using T. gondii antigens, or OVA and keyhole limpet hemocyanin in complete Freund’s adjuvant, and infection of MyD88–/– mice with L. major failed to induce Th1 cell responses. Rather, CD4+ T cells responded to antigen re-stimulation with the production of Th2 cytokines. The observed Th2 cell differentiation was most likely a consequence of highly impaired IL-12 production observed in MyD88-deficient mice (8,1012).

Here, we use the mouse L. monocytogenes infection model to analyze the role of TLR signaling in acquired immunity against L. monocytogenes. During the initial phase of infection, L. monocytogenes is controlled by innate immune mechanisms. Although these mechanisms initially restrict listerial growth, efficient control and ultimate eradication of L. monocytogenes is achieved by the Listeria-specific T cell response. While both CD4+ and CD8+ T cells participate in immunity against primary infection, CD8+ T cells are central for protection against re-infection (1316). Using mice deficient in TLR2, TLR4 or MyD88, we demonstrate the generation of Listeria-specific CD4+ Th1 and CD8+ T cells in the absence of TLR2, TLR4 or MyD88. Upon re-infection, these mice are protected against an otherwise lethal L. monocytogenes inoculum. Thus, TLR2-, TLR4- and MyD88-dependent signals are neither essential for the generation of CD4+ Th1 and CD8+ T cells nor for the protection mediated by these cells during re-infection.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies
Rat IgG antibodies, anti-CD16/CD32 mAb (clone 2.4G2), anti-IFN-{gamma} mAb (R4-6A2, rat IgG1), anti-CD8{alpha} mAb (YTS169), anti-CD4 mAb (YTS191.1) and anti-CD62L mAb (Mel-14), were purified from rat serum or hybridoma supernatants with Protein G–Sepharose. Antibodies were Cy5- or FITC-conjugated according to standard protocols. The FITC-conjugated rat IgG1 isotype control mAb (clone R3-34) was purchased from PharMingen (San Diego, CA).

Mice and L. monocytogenes infection
C57BL/6 were purchased from the Federal Institute for Health Protection of Consumers and Veterinary Medicine (Berlin, Germany) and bred in our facilities. TLR2–/–, TLR4–/– and MyD88–/– mice back-crossed onto the C57BL/6 background were kindly provided by Dr Shizuo Akira and bred in our facilities (1719). All animal experiments were conducted according to the German animal protection law. Mice were infected with a L. monocytogenes strain recombinant for a secreted form of OVA (LmOVA) kindly provided by Dr Hao Shen (20). Bacteria were grown overnight in tryptic soy broth (TSB), washed twice in PBS, aliquoted in PBS/10% glycerol and stored at –80°C. Aliquots were thawed and bacterial titers were determined by plating serial dilutions on TSB agar plates. For i.v. infection, bacteria were appropriately diluted and injected in a volume of 200 µl PBS into the lateral tail vein. In indicated experiments, mice were injected 2 days post-infection with 2 mg ampicillin in PBS i.p. and, for the following 5 days, mice were kept with 2 mg/ml ampicillin in the drinking water (21). For determination of bacterial burdens in organs, mice were killed, organs were homogenized in PBS and serial dilutions of homogenates were plated on TSB agar. Colonies were counted after 24 h incubation at 37°C.

In vitro re-stimulation of cells and flow cytometric determination of cytokine expression
Spleens were removed and single-cell suspensions were prepared using an iron mesh sieve. Red blood cells were lysed, and spleen cells were washed twice with RPMI 1640 medium supplemented with glutamine, sodium pyruvate, ß-mercaptoethanol, penicillin, streptomycin and 10% heat-inactivated FCS (complete RPMI medium). For the determination of cytokine expression, 4 x 106 cells were cultured in a volume of 1 ml complete RPMI medium. Cells were stimulated for 5 h with 10–6 M of the peptides listeriolysin O amino acids 190–201 (LLO190–201, NEKYAQAYPNVS) or OVA257–264 (SIINFEKL) (20,22). During the final 4 h of culture, 10 µg/ml Brefeldin A was added. Cultured cells were washed, and incubated for 10 min with rat IgG antibodies and anti-CD16/CD32 mAb to block non-specific antibody binding. Subsequently, cells were stained with Cy5-conjugated anti-CD4 mAb or anti-CD8{alpha} mAb, and after 30 min on ice cells were washed with PBS and fixed for 20 min at room temperature with PBS/4% paraformaldehyde. Cells were washed with PBS/0.1% BSA, permeabilized with PBS/0.1% BSA/0.5% saponin, and incubated in this buffer with rat IgG antibodies and anti-CD16/CD32 mAb. After 5 min, FITC-conjugated anti-IFN-{gamma} mAb or isotype control mAb were added. After a further 20 min at room temperature, cells were washed with PBS and fixed with PBS/1% paraformaldehyde. Cells were analyzed using a FACSCalibur and CellQuest software (Becton Dickinson, Mountain View, CA) (14).

Generation of MHC class I tetramers and staining of cells with tetramers
Modified forms of the full-length cDNA of H-2Kb and human ß2-microglobulin were kindly provided by Dr Dirk Busch. H-2Kb/OVA257–264 tetramers were produced as described (13,23). For flow cytometry analysis, 2 x 106 cells were incubated for 15 min at 4°C with rat IgG antibodies, anti-CD16/CD32 mAb and streptavidin (Molecular Probes, Eugene, OR) in PBS/0.5% BSA/0.01% sodium azide. Cells were then stained for 60 min at 4°C with Cy5-conjugated anti-CD8{alpha} mAb, FITC-conjugated anti-CD62L mAb and phycoerythrin-conjugated MHC class I–OVA257–264 tetramers. Subsequently, cells were washed with PBS/0.5% BSA/0.01% sodium azide and diluted in PBS. Propidium iodide was added prior to four-color flow cytometry analysis.

Statistical analysis
Statistical significance of results was determined with the statistics program included in the GraphPad Prism software (San Diego, CA). Bacterial titers were analyzed with the Mann–Whitney test and frequencies and numbers of tetramer-positive or cytokine-expressing cells with the unpaired Student’s t-test. P < 0.05 was considered as a statistically significant difference.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Abrogation of L. monocytogenes infection does not impair Listeria-specific CD4+ and CD8+ T cell responses
Infection of mice with L. monocytogenes induces strong CD4+ and CD8+ T cell responses (13,14). To directly analyze Listeria-specific H-2b restricted T cell responses ex vivo, we used a L. monocytogenes strain recombinant for OVA (20). Upon infection with LmOVA, C57BL/6 mice mount a CD8+ T cell response against the peptide OVA257–264 and a CD4+ Th1 cell response against the peptide LLO190–201 (14,22,24). These T cell responses can be detected with OVA257–264/H-2Kb tetramers or with intracellular IFN-{gamma} staining after short-term in vitro stimulation with the peptides OVA257–264 or LLO190–201. In some mouse strains, high susceptibility against L. monocytogenes infection precludes direct analysis of T cell responses. To circumvent this problem, we used a protocol in which mice were infected and 2 days later treated with ampicillin to clear Listeria from the mice (21). Mercado et al. recently demonstrated that removal of L. monocytogenes with antibiotics as early as 24 h after infection did not impair the magnitude and quality of the Listeria-specific CD8+ T cell response, indicating that a short period of antigen contact is sufficient to induce a CD8+ T cell response (21). Our results are consistent with this observation (Fig. 1). Treatment of mice with ampicillin did not significantly change the OVA257–264-specific CD8+ T cell response in mice infected with LmOVA. Furthermore, the analysis of the LLO190–201-specific CD4+ T cell response revealed that chemotherapy did not impair the generation of the CD4+ Th1 responses either (Fig. 1).



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Fig. 1. Treatment of L. monocytogenes-infected mice with antibiotics does not significantly impair CD4+ and CD8+ T cell responses. C57BL/6 mice were i.v. infected with 4 x 103 LmOVA. Two days post-infection, mice received 2 mg ampicillin i.p. and were kept for a further 5 days on 2 mg/ml ampicillin in the drinking water. After 10 days, mice were killed, and spleen cells were directly analyzed with OVA257–264 tetramers or re-stimulated in vitro with LLO190–201 and examined for IFN-{gamma} production by intracellular cytokine staining. The upper panel shows the result of OVA257–264 tetramer staining from CD8-gated cells; the lower panel, IFN-{gamma} production of CD4-gated cells after incubation with or without LLO190–201. Numbers give means of percentage values ± SD of positive CD8+ or CD4+ T cells from three individually analyzed mice per group and are representative of two independent experiments. Intracellular staining with FITC-conjugated isotype control mAb always resulted in <0.05% positive cells (not shown).

 
TLR2–/– and MyD88–/– mice mount Listeria-specific T cell responses
TLR2 recognizes cell wall components of Gram-positive bacteria such as lipoteichoic acids and peptidoglycans (3). As a consequence, TLR2–/– mice produce reduced amounts of IL-12 and IFN-{gamma} following infection with L. monocytogenes and TLR2-deficient macrophages are impaired in their in vitro responses to live and heat-killed Listeria (5,6). Despite these defects, TLR2–/– mice show enhanced susceptibility only after intracerebral, but not after i.v. or i.p. infection (5,6,25). MyD88–/– mice and MyD88-deficient macrophages display profound defects in response to L. monocytogenes, and in contrast to TLR2–/– mice, MyD88–/– mice are highly susceptible to L. monocytogenes infection (5,6). To determine whether impaired responses of TLR2–/– and MyD88–/– mice to L. monocytogenes influence the generation of Listeria-specific T cells, mice from both strains were infected with LmOVA. As controls, TLR4–/– and wild-type C57BL/6 mice were infected. Two days post-infection, all mice were treated with ampicillin and at 10 days post-infection spleens of mice were analyzed for Listeria-specific T cell responses (Figs 2 and 3). Determination of OVA257–264-specific CD8+ T cells with tetramers revealed that neither the MyD88 nor the TLR2 deficiency impaired the Listeria-specific CD8+ T cell response significantly (Fig. 2). After short-term in vitro re-stimulation with OVA257–264 and intracellular IFN-{gamma} staining, we observed slightly reduced frequencies of OVA257–264-specific CD8+ T cells compared to the determination with tetramers (Fig. 3A and B). However, in all mouse strains, similar frequencies of Listeria-specific T cells were detected, indicating that the OVA257–264-specific CD8+ T cells were functional effector cells, at least in terms of IFN-{gamma} production. The LLO190–201-specific CD4+ T cell response showed a similar pattern. Although there was some variation in the strength of response in the analyzed mouse strains, all mice were able to elicit a Listeria-specific CD4+ Th1 response (Fig. 3C and D).



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Fig. 2. TLR2–/–, TLR4–/– and MyD88–/– mice mount normal CD8+ T cell responses to L. monocytogenes. TLR2–/–, TLR4–/–, MyD88–/– and wild-type control mice were i.v. infected with 4 x 103 LmOVA. Two days post-infection, mice received 2 mg ampicillin i.p. and were kept for a further 5 days on 2 mg/ml ampicillin in the drinking water. On day 10 post-infection, mice were killed and spleen cells were analyzed with OVA257–264 tetramers. (A) Percentage values of OVA257–264 tetramer+ cells of CD8+ T cells. (B) Total number of OVA257–264 tetramer+ CD8+ T cells per spleen (means ± SD of three individually analyzed mice per group, representative of two independent experiments). In (A) and (B), frequencies and numbers of Listeria-specific CD8+ T cells from spleens of infected TLR2–/–, TLR4–/– and MyD88–/– mice were not significantly different to those from infected wild-type mice (P > 0.05).

 


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Fig. 3. TLR2–/–, TLR4–/– and MyD88–/– mice mount normal CD4+ and CD8+ T cell responses to L. monocytogenes. TLR2–/–, TLR4–/–, MyD88–/– and wild-type control mice were infected and treated as described in the legend of Fig. 2. Ten days post-infection, spleen cells were re-stimulated with OVA257–264 or LLO190–201 and analyzed for IFN-{gamma}-producing CD8+ (A and B) or CD4+ T cells (C and D) respectively by intracellular IFN-{gamma} staining. (A and C) Percentage values of IFN-{gamma}+ cells of CD8+ and CD4+ T cells, and (C and D) total numbers of IFN-{gamma}+CD8+ and IFN-{gamma}+CD4+ T cells/spleen. Background frequencies (<0.05%) and numbers of IFN-{gamma}-producing cells determined in cultures without peptides were subtracted from frequencies and numbers derived from cultures with peptides. Numbers and percentage values represent means ± SD of three individually analyzed mice per group and are representative of two independent experiments. In (A–D), frequencies and numbers of Listeria-specific CD4+ and CD8+ T cells in spleens of infected TLR2–/–, TLR4–/– and MyD88–/– mice were not significantly different to those from infected wild-type mice (P > 0.05).

 
TLR2–/–, TLR4–/– and MyD88–/– mice are protected against secondary L. monocytogenes infection
Since TLR2–/–, TLR4–/– and MyD88–/– mice generated normal T cell responses against L. monocytogenes during primary infection, we asked whether these T cells protected against secondary infection. TLR2–/–, TLR4–/–, MyD88–/– and wild-type control mice were infected with LmOVA. Two days after infection, mice were treated with ampicillin to clear the infection. After 4 weeks, mice were re-infected with a high dose of LmOVA, and 2 days later, Listeria titers in the spleens were analyzed. Additionally, naive mice of all four strains were infected with the same LmOVA dose and analyzed in parallel (Fig. 4). Comparison of Listeria titers in primary and secondary infected mice revealed significantly reduced titers in secondary infected mice from all strains analyzed. In all experiments, we observed the highest level of protection in re-infected wild-type mice. However, differences in titers from re-infected wild-type mice and re-infected TLR2–/–, TLR4–/– or MyD88–/– mice never reached a significant level (P values always >0.05). Our results indicate that Listeria-specific T cells acquired during primary infection protected against secondary infection, and that this protection was independent of TLR2, TLR4 and MyD88. Thus, the acquired immune system can control L. monocytogenes-infection in the absence of TLR2- and MyD88-mediated signals.



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Fig. 4. L. monocytogenes titers in spleens of primary and secondary infected TLR2–/–, TLR4–/– and MyD88–/– mice. TLR2–/–, TLR4–/–, MyD88–/– and wild-type control mice were i.v. infected with 4 x 103 LmOVA. Two days post-infection, mice received 2 mg ampicillin i.p. and were kept for a further 5 days on 2 mg/ml ampicillin in the drinking water. Thirty days after primary infection, mice were secondary i.v. infected with 105 LmOVA (filled symbols). In parallel, naive mice received 105 LmOVA (open symbols). Two days later, mice were killed and bacterial titers in spleens were determined. Results are representative of two independent experiments. For all mouse strains, titers in secondary infected mice were significantly lower than in primary infected mice (P < 0.05).

 
T cell responses in TLR2–/–, TLR4–/– and MyD88–/– mice after secondary L. monocytogenes infection
After secondary infection with LmOVA, we also determined the magnitude of the T cell responses in TLR2–/–, TLR4–/–, MyD88–/– and wild-type mice. Six days after secondary infection, spleens of mice were analyzed using OVA257–264 tetramers and intracellular IFN-{gamma}-staining after in vitro re-stimulation with LLO190–201 (Fig. 5). In all mouse strains, we detected enlarged populations of Listeria-specific CD4+ and CD8+ T cells. Compared to the primary responses (Figs 2 and 3), T cell populations were enlarged and responses were accelerated, indicating that TLR2–/–, TLR4–/– and MyD88–/– mice were able to mount normal memory T cell responses.



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Fig. 5. TLR2–/–, TLR4–/– and MyD88–/– mice mount normal secondary T cell responses to L. monocytogenes. TLR2–/–, TLR4–/–, MyD88–/– and wild-type control mice were i.v. infected with 4 x 103 LmOVA. Two days post-infection, mice received 2 mg ampicillin i.p. and were kept for a further 5 days on 2 mg/ml ampicillin in the drinking water. Thirty days after primary infection, mice were secondary i.v. infected with 105 LmOVA. After a further 6 days, CD8+ and CD4+ T cell responses were analyzed with OVA257–264 tetramers (A) or with intracellular cytokine staining after re-stimulation with LLO190–201 peptide (B) respectively. Numbers represent means ± SD of three individually analyzed mice per group. In (A) and (B), numbers of Listeria-specific CD4+ and CD8+ T cells from spleens of infected TLR2–/–, TLR4–/– and MyD88–/– mice were not significantly different to numbers from infected wild-type mice (P > 0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our results allow two central conclusions: (i) TLR2, TLR4 and MyD88 are not required for the generation of CD4+ Th1 and CD8+ T cell responses against L. monocytogenes, and (ii) these responses are sufficient to control a subsequent L. monocytogenes infection in the absence of TLR2-, TLR4- and MyD88-mediated signals.

In the case of TLR4, this result may not be a surprise, since TLR4 is mostly required to control Gram-negative bacteria and appears to play only a minor role in the defense against L. monocytogenes. TLR4–/– mice demonstrate normal control of L. monocytogenes and only marginally impaired production of inflammatory cytokines (6). For TLR2 and MyD88 the situation is different. As the central TLR recognizing products of Gram-positive bacteria such as peptidoglycans and lipoteichoic acids, TLR2 should be of major importance in immunity against L. monocytogenes. TLR2–/– mice indeed show reduced production of inflammatory cytokines, e.g. IL-12 and TNF-{alpha}, upon L. monocytogenes infection (5,6). MyD88, as a central element of the signal transduction cascade of all TLR as well as the IL-1 receptor family, plays an even more pivotal role in the induction of immune responses against L. monocytogenes infection. MyD88–/– mice are highly susceptible to L. monocytogenes and display profound defects in the generation of inflammatory cytokines upon infection (5,6). Overall, these results indicate that TLR2 and particularly MyD88-mediated signals from TLR are important for recognition of L. monocytogenes by the innate immune system and for initiation of early immune defense mechanisms. It was, therefore, surprising that generation of specific CD4+ Th1 and CD8+ T cell responses against L. monocytogenes were not impaired in TLR2–/– and MyD88–/– mice. A recently published study by Way et al. is consistent with our result (26). In this study, the T cell response of MyD88–/– mice to a highly attenuated actA-deficient L. monocytogenes strain was analyzed. MyD88–/– mice mounted a normal CD8+ T cell response and only a modestly impaired CD4+ T cell response against the attenuated L. monocytogenes strain. Furthermore, MyD88–/– mice infected with the attenuated strain were protected against a challenge with a wild-type strain of L. monocytogenes and transfer of CD8+ T cells from these mice resulted in protection of naive MyD88–/– mice against L. monocytogenes infection (26). Overall, our results and the results from Way et al. contrast with observations from other immunization and infection studies, where MyD88–/– mice failed to generate Th1 responses, but rather developed aberrant Th2 responses (8,1012).

The initial events and requirements for the generation of CD4+ and CD8+ T cell responses against L. monocytogenes are ill defined. However, we assume that signals from PAMP receptors must be involved in the activation and maturation of professional antigen-presenting cells to allow priming of Listeria-specific T cells and in the generation of inflammatory cytokines, which drive CD4+ T cells into the Th1 linage. In this context, our results imply TLR2- and MyD88-independent receptors, which recognize L. monocytogenes and initiate T cell priming and differentiation. Indeed, there is indirect evidence for such receptors (5,6). Infection of TLR2–/– mice with L. monocytogenes induces reduced, but still measurable, serum levels of IL-12 and IFN-{gamma}. TLR2–/– macrophages produce low amounts of TNF-{alpha}, IL-12 and NO radicals in vitro (5,6). Even MyD88–/– mice are not completely insensitive against L. monocytogenes infection, and respond with increased serum levels of TNF-{alpha}, NO radicals and IFN-{gamma} (although IFN-{gamma} levels are highly reduced compared to wild-type controls) (5). Finally, after in vitro activation with IFN-{gamma}, macrophages of TLR2–/– and MyD88–/– mice express listeriocidal activity similar to wild-type macrophages (5). This observation further underlines the existence of TLR2- and MyD88-independent receptors for L. monocytogenes in macrophages, and offers a mechanism for protection during secondary L. monocytogenes infection.

The identity of the alternative receptor(s) for L. monocytogenes is completely unclear. In TLR2–/– mice, it is likely that listerial compounds are recognized by other TLR such as TLR5 (flagellin) or TLR9 (unmethylated CpG motifs) (3). The situation is different for MyD88–/– mice. Although MyD88-independent TLR signaling has been described recently, so far evidence for such signaling pathways only exists for TLR3 and TLR4. It is most likely that both receptors play a marginal role in the recognition of L. monocytogenes and even for these receptors MyD88-independent pathways can only partially compensate the MyD88-mediated signals (3,4). We, therefore, assume that other, TLR-independent receptors recognize L. monocytogenes and initiate T cell priming. Candidates include lectin-domain-containing receptors like the mannose receptor or DC205, scavenger receptors, complement receptors and Fc receptors recognizing L. monocytogenes opsonized with complement or natural antibodies respectively, or intracellular receptors such as members of the NOD protein family [reviewed in (2)]. Of course, our findings do not exclude participation of TLR- or MyD88-dependent signaling pathways in immunocompetent mice. As known from other systems, compensatory mechanisms assume functions in the absence of mechanisms which perform the first line of defense (27). It is a major advantage of knockout systems to uncover such compensatory lines of defense. Infection of MyD88–/– mice with L. monocytogenes offers a valuable experimental model to elucidate the nature and function of such alternative PAMP receptors.


    Acknowledgements
 
We thank Drs Shizuo Akira, Dirk Busch and Hao Shen for providing mice and reagents. We further acknowledge the help of Manuela Stäber, Daniela Groine-Triebkorn, Dr Robert Hurwitz and Dr Uwe Klemm for help with the production of tetramers, purification of antibodies and for typing of mice. S. H. E. K. acknowledges financial support from the Deutsche Forschungsgemeinschaft (Priority Program ‘Novel vaccination strategies’).


    Abbreviations
 
LLO—listeriolysin O

LmOVA—ovalbumin-secreting strain of Listeria monocytogenes

MyD88—myeloid differentiation factor 88

OVA—ovalbumin

PAMP—pathogen-associated molecular pattern

TLR—Toll-like receptor

TNF—tumor necrosis factor

TSB—tryptic soy broth


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Janeway, C. A., Jr and Medzhitov, R. 2002. Innate immune recognition. Annu. Rev. Immunol. 20:197.[CrossRef][ISI][Medline]
  2. Underhill, D. M. 2003. Toll-like receptors: networking for success. Eur. J. Immunol. 33:1767.[CrossRef][ISI][Medline]
  3. Takeda, K., Kaisho, T. and Akira, S. 2003. Toll-like receptors. Annu. Rev. Immunol. 21:335.[CrossRef][ISI][Medline]
  4. Barton, G. M. and Medzhitov, R. 2003. Toll-like receptor signaling pathways. Science 300:1524.[Abstract/Free Full Text]
  5. Edelson, B. T. and Unanue, E. R. 2002. MyD88-dependent but Toll-like receptor 2-independent innate immunity to Listeria: no role for either in macrophage listericidal activity. J. Immunol. 169:3869.[Abstract/Free Full Text]
  6. Seki, E., Tsutsui, H., Tsuji, N. M., Hayashi, N., Adachi, K., Nakano, H., Futatsugi-Yumikura, S., Takeuchi, O., Hoshino, K., Akira, S., Fujimoto, J. and Nakanishi, K. 2002. Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice. J. Immunol. 169:3863.[Abstract/Free Full Text]
  7. Takeuchi, O., Hoshino, K. and Akira, S. 2000. TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection. J. Immunol. 165:5392.[Abstract/Free Full Text]
  8. Muraille, E., De Trez, C., Brait, M., De Baetselier, P., Leo, O. and Carlier, Y. 2003. Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response. J. Immunol. 170:4237.[Abstract/Free Full Text]
  9. Scanga, C. A., Aliberti, J., Jankovic, D., Tilloy, F., Bennouna, S., Denkers, E. Y., Medzhitov, R. and Sher, A. 2002. MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J. Immunol. 168:5997.[Abstract/Free Full Text]
  10. Schnare, M., Barton, G. M., Holt, A. C., Takeda, K., Akira, S. and Medzhitov, R. 2001. Toll-like receptors control activation of adaptive immune responses. Nat. Immunol. 2:947.[CrossRef][ISI][Medline]
  11. Jankovic, D., Kullberg, M. C., Hieny, S., Caspar, P., Collazo, C. M. and Sher, A. 2002. In the absence of IL-12, CD4+ T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10/– setting. Immunity 16:429.[ISI][Medline]
  12. Kaisho, T., Hoshino, K., Iwabe, T., Takeuchi, O., Yasui, T. and Akira, S. 2002. Endotoxin can induce MyD88-deficient dendritic cells to support Th2 cell differentiation. Int. Immunol. 14:695.[Abstract/Free Full Text]
  13. Busch, D. H., Pilip, I. M., Vijh, S. and Pamer, E. G. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8:353.[ISI][Medline]
  14. Kursar, M., Bonhagen, K., Köhler, A., Kamradt, T., Kaufmann, S. H. E. and Mittrücker, H.-W. 2002. Organ-specific CD4+ T cell response during Listeria monocytogenes infection. J. Immunol. 168:6382.[Abstract/Free Full Text]
  15. Kaufmann, S. H. E. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129.[CrossRef][ISI][Medline]
  16. Mittrücker, H.-W., Köhler, A. and Kaufmann, S. H. E. 2000. Substantial in vivo proliferation of CD4+ and CD8+ T lymphocytes during secondary Listeria monocytogenes infection. Eur. J. Immunol. 30:1053.[CrossRef][ISI][Medline]
  17. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K. and Akira, S. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443.[ISI][Medline]
  18. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K. and Akira, S. 1999. Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749.[Abstract/Free Full Text]
  19. Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K. and Akira, S. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143.[ISI][Medline]
  20. Foulds, K. E., Zenewicz, L. A., Shedlock, D. J., Jiang, J., Troy, A. E. and Shen, H. 2002. CD4 and CD8 T cells are intrinsically different in their proliferative responses. J. Immunol. 168:1528.[Abstract/Free Full Text]
  21. Mercado, R., Vijh, S., Allen, S. E., Kerksiek, K., Pilip, I. M. and Pamer, E. G. 2000. Early programming of T cell populations responding to bacterial infection. J. Immunol. 165:6833.[Abstract/Free Full Text]
  22. Geginat, G., Schenk, S., Skoberne, M., Goebel, W. and Hof, H. 2001. A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes. J. Immunol. 166:1877.[Abstract/Free Full Text]
  23. Mittrücker, H.-W., Kursar, M., Köhler, A., Hurwitz, R. and Kaufmann, S. H. E. 2001. Role of CD28 for the generation and expansion of antigen-specific CD8+ T lymphocytes during infection with Listeria monocytogenes. J. Immunol. 167:5620.[Abstract/Free Full Text]
  24. Pope, C., Kim, S.-K., Marzo, A., Williams, K., Jiang, J., Shen, H. and Lefrancois, L. 2001. Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J. Immunol. 166:3402.[Abstract/Free Full Text]
  25. Echchannaoui, H., Frei, K., Schnell, C., Leib, S. L., Zimmerli, W. and Landmann, R. 2002. Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation. J. Infect. Dis. 186:798.[CrossRef][ISI][Medline]
  26. Way, S. S., Kollmann, T. R. Hajjar, A. M. and Wilson, C. B. 2003. Protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88. J. Immunol. 171:533.[Abstract/Free Full Text]
  27. Kaufmann, S. H. E. 1994. Bacterial and protozoal infections in genetically disrupted mice. Curr. Opin. Immunol. 6:518.[CrossRef][ISI][Medline]




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