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The Journal of Immunology, 2001, 166: 466-472.
Copyright © 2001 by The American Association of Immunologists

Listeria monocytogenes Modulates Macrophage Cytokine Responses Through STAT Serine Phosphorylation and the Induction of Suppressor of Cytokine Signaling 31

Dagmar Stoiber*, Silvia Stockinger*, Peter Steinlein{dagger}, Jan Kovarik{ddagger} and Thomas Decker2,*

* Institute of Microbiology and Genetics, and {dagger} Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria; and {ddagger} Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophage activation as part of natural resistance to infection is caused by stimulation with IFN-{gamma} and by the invading microorganisms or microbial products. Infection of macrophages with the Gram-positive bacterium Listeria monocytogenes for short periods before activation with IFN-{gamma} increased the phosphorylation of transcription factor STAT1 at S727 and thereby the expression of IFN-{gamma}-induced genes. By contrast, persistent infection with viable bacteria or treatment with heat-killed Listeria diminished IFN-{gamma}-stimulated transcription and the phosphorylation of STAT1 at Y701. Decreased IFN-{gamma} signaling correlated with the induction of suppressor of cytokine signaling 3 (SOCS3) mRNA and protein. Contrasting our previous findings with LPS, maximal synthesis of SOCS3 required both the immediate signals from Listeria receptors on the cell surface and the activity of a polypeptide secreted in response to bacterial infection. SOCS3 induction by the secreted protein could not be blocked by neutralizing Abs to IL-10 and it did not require the presence of STAT1. Consistent with the induction of SOCS3 activity, Listeria also inhibited activation of STAT5 by GM-CSF. The p38 mitogen-activated protein kinase was rapidly activated upon infection of macrophages with L. monocytogenes. Inhibition of p38 mitogen-activated protein kinase with the pyridinyl imidazol SB203580 abrogated both STAT1 S727 phosphorylation and the expression of SOCS3. The data suggest that STAT1 serine kinase and SOCS3 activity are hallmarks of immediate and delayed phases of influence by bacterial signals on signal transduction in response to IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages are important effector cells of the innate immune system. The complete immunological activation of these cells produces a maximal capacity to ingest and kill microbes, synthesize membrane receptors, and secrete molecules that function in a proinflammatory immune response. Full activation depends upon two distinct stimuli. One signal is provided by the Th1 cytokine IFN-{gamma}, whereas the second is most often provided by components of microbial cell walls or membranes (1, 2). IFN-{gamma} influences nuclear gene expression by binding to a heterooligomeric, class II cytokine receptor and activating the receptor-associated Janus tyrosine kinases JAK13 and JAK2. These phosphorylate the STAT1 at Y701 and cause its dimerization and translocation to the nucleus (3, 4). STAT1 dimers then bind to their cognate promoter DNA, the IFN-{gamma} activation site (GAS), and activate transcription (5). To act with high efficiency as a transcription factor, STAT1 must also be phosphorylated at S727, which lies within a potential mitogen-activated protein kinase (MAPK) consensus motif at the C terminus, a putative transactivation domain (6, 7). At present, the kinase(s) phosphorylating S727 is/are not clearly defined, but the p38 MAPK was shown to be required for LPS or stress-induced phosphorylation of STAT1 at S727 (8).

Invading microorganisms transduce signals through a variety of pattern recognition receptors (reviewed in Ref. 9). Among these are the recently identified Toll-like receptors (TLRs) that discriminate between different microbial pathogens or their products (10, 11). Whereas TLR4 is the main protein involved in recognizing Gram-negative bacteria and LPS, TLR2 is a key component in responses to other types of microbial pathogens, such as yeast and Gram-positive bacteria (12, 13, 14, 15). LPS/TLR4 stimulate gene expression predominantly through the activation of transcription factor NF-{kappa}B (16). However, LPS also directly influences the output of the JAK-STAT signal transduction pathway in a time-dependent manner. Although short time exposure of macrophages to LPS increases the activation of STAT1 via the activation of a STAT1 S727 kinase (8, 17), prolonged treatment with LPS causes down-regulation of IFN-{gamma}-mediated STAT1 tyrosine phosphorylation and transcription. This inhibitory effect is due to an immediate and direct signal of LPS to the suppressor of cytokine signaling 3 (socs3) gene (18). The SOCS family of proteins, also known as cytokine-inducible Src homology 2 domain-containing proteins, JAK-binding proteins, or STAT-induced STAT inhibitors (SSI) (19, 20, 21), was recently described as feedback inhibitors of cytokine signaling pathways employing JAKs and STATs (reviewed in Ref. 22). However, as demonstrated for LPS or TNF-{alpha} (17, 23, 24), SOCS also mediate negative cross-talk between JAK-STAT pathways and other signaling routes.

The goal of our study was to investigate whether the induction of SOCS3 and/or the activation of the STAT1 serine kinase is unique to the LPS response or whether it is a general property of pattern recognition receptors. Therefore, we studied the impact of infection with Listeria monocytogenes on the macrophages’ response to IFN-{gamma}. L. monocytogenes is a facultative intracellular Gram-positive pathogen that replicates in the cytosol of mammalian cells (reviewed in Refs. 25, 26). Once internalized, Listeria are able to lyse the vacuolar membrane due to the activity of a potent pore-forming toxin, listeriolysin O (LLO). Mutants that do not express LLO are avirulent in mouse, demonstrating that escape of the bacteria from the phagosome to the cytosol is critical for the establishment of an infection (27, 28, 29). We report the phosphorylation of STAT1 S727 in response to Listeria infection and show that Listeria can enhance IFN-{gamma} signaling. Similar to LPS, the continuous presence of Listeria reduces signal transduction in response to IFN-{gamma} through the induced synthesis of SOCS3. However, unlike LPS, SOCS3 induction by Listeria occurs in part through a secondary response requiring the activity of a secreted cytokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Bac1.2F5 macrophages (30), or C11 cells, a clone of Bac1.2F5 transfected with a STAT-dependent luciferase gene (17), were maintained in MEM-{alpha} medium containing 10% FCS and 20% L cell-conditioned medium as a source of CSF-1. Bone marrow-derived macrophages were derived, as described previously (31), after isolation of bone marrow from femurs of wild-type or STAT1-deficient mice. STAT1-deficient mice were provided by Dr. D. E. Levy, New York University School of Medicine (New York, NY).

Bacterial strains, cytokines, and drugs

L. monocytogenes (Bof 343) and the isogenic, nonhemolytic mutant (Bof 415), obtained from Pascale Cossart (Institute Pasteur, Paris, France), were cultured in brain heart infusion broth (32). Heat-killed cells of L. monocytogenes (hkL) were prepared by incubating the viable bacterial suspension at 70°C for 10 min. For stimulation of cells, IFN-{gamma} (provided by G. Adolf, Boehringer Ingelheim, Vienna, Austria) was used at a concentration of 10 ng/ml. GM-CSF was prepared from the GM-CSF-producing cell line X6310 GM-CSF that was grown in RPMI medium supplemented with 10% FCS for 2 days, and the supernatant was collected and sterile filtered and used at a concentration of 1:100. LPS (Sigma, St. Louis, MO) was from Salmonella minnesota and used at a concentration of 1 µg/ml. The p38 MAPK inhibitor SB203580 was provided by Ken Murray (SmithKline Beecham, Philadelphia, PA) and was used at 10 µM. The drug was added 30 min before further treatment.

Cell infection

Bac1.2F5 macrophages were infected with L. monocytogenes wild-type, mutant (both derived from overnight culture), or heat-killed bacteria at multiplicity of infection of 10:1 and incubated for 1 h at 37°C in a humidified 5% CO2 atmosphere. Extracellular bacteria were removed by washing with PBS and killed with gentamicin-containing medium (final concentration 50 µg/ml). After another 60 min, medium was changed to medium containing 10 µg/ml gentamicin. This concentration was shown not to affect survival of intracellular Listeria during the period of treatment (33).

Antibodies

The antisera to the STAT1 C terminus and to phospho-S727-STAT1 have recently been described (17). Rabbit antiserum to Y701-phosphorylated STAT1 was purchased from New England Biolabs (Beverly, MA) and used at a dilution of 1:1000. Rabbit anti-SOCS3 antiserum was produced by immunization with a GST fusion protein of SOCS3 (the plasmid was provided by Jim Johnston, DNAX Research Institute, Palo Alto, CA). It was used at a 1:1000 dilution in Western blots. A mAb to SOCS3 was derived from spleen cells of mice after immunization with the GST-SOCS3 fusion protein by standard techniques. Hybridoma supernatants were screened for specific Ab by ELISA with GST-SOCS3-coated wells and subsequently by Western blotting of extracts from SOCS3-transfected 293 cells. Undiluted hybridoma supernatants were used for the experiment shown in Fig. 5GoC. A polyclonal Ab against Y694/Y699-phosphorylated STAT5a/b was purchased from Upstate Biotechnology (Lake Placid, NY). The antiserum specific for the STAT5a isoform (and its use in Western blot experiments) was recently described (34). Neutralizing Abs to IL-10 were purchased from Strathmann Biotech (Hannover, Germany). They were used at a dilution of 1:200, which neutralizes 2 ng/ml of IL-10. The amount secreted by LPS-treated monocytes or macrophages into the culture medium was reported to be at least 4-fold lower (35, 36). Abs specific for the phosphorylated form of p38 MAPK, or against p38 MAPK, were bought from New England Biolabs. Pan-extracellular signal-related kinase mAbs were purchased from Transduction Laboratories (Lexington, KY) and used at a dilution of 1:2000 in Western blots. For FACS analysis, a biotin-labeled rat mAb recognizing the {alpha}-chain of the murine IFN-{gamma} receptor was obtained from PharMingen (San Diego, CA). PE-labeled streptavidin was used as a secondary reagent. Rabbit antisera to JAK1 and JAK2 kinases were a gift from Dr. Andrew Zimiecki (Laboratory for Clinical and Experimental Cancer Research, Bern, Switzerland). They were used at 1:100 dilution for immuno-precipitation and 1:1000 dilution for Western blots. Polyclonal Abs against inhibitory protein that dissociates from NF-{kappa}B (I-{kappa}B{alpha}) were used in Western blots at a dilution of 1:1000.



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  		FIGURE 5. Listeria infection causes expression of SOCS3 mRNA and protein in macrophages. A, Bac1.2F5 cells were infected with Listeria, as shown. Where indicated, cycloheximide (50 µg/ml) or neutralizing Ab against murine IL-10 (1:200) was present. Total RNA was extracted and analyzed for SOCS3 RNA by Northern blotting. B, Extracts from Listeria wild-type (WT) or hkL-treated macrophages were analyzed by Western blotting using SOCS3 antiserum.

 
Immunoprecipitation, Western blot

We have recently described protocols for these procedures (17).

EMSA

An end-labeled, double-stranded oligonucleotide corresponding to the GAS sequence of the {beta}-casein promoter was used. The assay was performed with whole cell extracts, as recently described (37, 38).

Luciferase assay

Extracts from C11 macrophages were assayed for luciferase activity according to standard procedures (39). Results are stated as inducibility, the ratio obtained by dividing cpm light emission from IFN-{gamma} and/or L. monocytogenes-treated and unstimulated cells.

Northern blot

An amount of 15 µg of total RNA from Bac1.2F5 cells was separated on agarose gels and blotted to membrane using standard procedures. The blots were probed using SOCS3 cDNA labeled by random priming.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
L. monocytogenes infection activates the STAT1 S727 kinase

In macrophages, phosphorylation of STAT1 S727 occurs in response to LPS, the outer membrane constituent of Gram-negative bacteria (17). To determine whether Gram-positive bacteria that signal through a distinct TLR family member cause STAT1 S727 phosphorylation, we infected macrophages with L. monocytogenes. The experiment shown in Fig. 1GoA demonstrates rapid phosphorylation of STAT1 S727 in response to infection with Listeria, which persisted throughout the tested period. Therefore, like LPS, infection with Gram-positive bacteria targets STAT1 at S727. IFN-{gamma} also caused phosphorylation of STAT1 at S727, and the effects of Listeria and IFN-{gamma} in phosphorylating STAT1 were approximately additive.



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  		FIGURE 1. Induction of STAT1 S727 phosphorylation in Bac1.2F5 macrophages in response to Listeria infection. Bac1.2F5 macrophages were infected with L. monocytogenes and/or IFN-{gamma} for 20 min, and whole cell extracts were prepared after the indicated periods. STAT1 phosphorylation and expression were determined in Western blots with appropriate Abs. A, Time course of STAT1 S727 phosphorylation after infection with L. monocytogenes with or without subsequent IFN-{gamma} treatment. B, Activation of p38 MAPK by L. monocytogenes. Macrophages were treated with hkL for the indicated periods, and p38 MAPK activation was determined by Western blot of cell lysates. The membrane was first incubated with a phosphospecific antiserum, then stripped and reprobed for the amount of p38 MAPK protein in each lane. C, Involvement of p38 MAPK in STAT1 S727 phosphorylation. The cells were stimulated with hkL or LPS for the indicated periods. SB203580 (10 µM) was present where indicated.

 
Bacterial infections cause activation of stress-activated MAPK. The two stress-activated paths targeting c-Jun kinases or the p38 MAPK isoenzymes can be distinguished using the pyridinyl imidazol drug SB203580, which at 10 µM specifically inhibits p38 MAPK{alpha} and {beta} both in vitro and in vivo (40, 41, 42). Infection with L. monocytogenes caused rapid activation of p38 MAPK (Fig. 1GoB). A potential role of p38 MAPK in phosphorylating STAT1 S727 was tested using SB203580 (Fig. 1GoC). The drug inhibited STAT1 S727 phosphorylation in response to hkL and LPS in macrophages by ~70%. This suggests a role for p38 MAPK{alpha}, because macrophages mainly express this isoform (43). However, macrophages also express the SB203580-insensitive p38 MAPK{delta}. Therefore, the incomplete suppression of STAT1 S727 phosphorylation by SB203580, which was also noted in the case of LPS (8), may result from activation of this p38 MAPK isoform, or be due to other SB203580-insensitive kinases participating in STAT1 phosphorylation.

Prolonged infection with Listeria inhibits STAT1 tyrosine phosphorylation

To investigate whether L. monocytogenes infection affects STAT1 tyrosine phosphorylation by IFN-{gamma}, we performed EMSA experiments. Infection of Bac1.2F5 macrophages (Fig. 2GoA, lanes 3–8), or bone marrow-derived macrophages (data not shown) with Listeria for more than 4 h strongly reduced the dimerization of STAT1 in response to a subsequent IFN-{gamma} stimulus. Furthermore, not only viable bacteria, but also hkL could significantly down-regulate STAT1 DNA-binding activity (lanes 9–14). Consistent with the EMSA results, pretreatment with viable wild-type or listeriolysin-deficient Listeria, or with hkL, reduced the tyrosine phosphorylation of STAT1 in response to IFN-{gamma}, as determined by Western blot with an antiserum against STAT1 phosphorylated on Y701 (Fig. 2GoB). Although some reduction of STAT1 protein expression was noted upon persistent Listeria infection or hkL treatment, control blots with pan-extracellular signal-related kinase antiserum showed that the reduced STAT1 tyrosine phosphorylation was not primarily caused by a decrease in the relative amount of cellular STAT1 protein. Furthermore, suppression of STAT1 tyrosine phosphorylation occurred at both saturating and limiting doses of IFN-{gamma} (Fig. 2GoC). Long exposures of our blots indicated that despite the ability of Listeria to down-regulate IFN-{gamma}-induced STAT1 tyrosine phosphorylation, a low level of STAT1 tyrosine phosphorylation was caused by stimulation with Listeria alone (data not shown).



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  		FIGURE 2. Effect of Listeria infection on STAT1 tyrosine phosphorylation in Bac1.2F5 macrophages. Cells were stimulated with IFN-{gamma} or Listeria alone, or a combination of both agents for the indicated periods. A, STAT1 DNA-binding activity in cell extracts was determined in an EMSA with a labeled STAT binding site (GAS) from the rat {beta}-casein promoter. Cells were either untreated (C, lane 1), stimulated with IFN-{gamma} alone for 20 min (lane 2), or infected with Listeria wild type (WT) (lanes 3–8) or hkL (lanes 9–14) for the indicated periods, followed by 20-min stimulation with IFN-{gamma}. B, Macrophage extracts after treatment with Listeria wild type (WT), listeriolysin mutant (LLO-), hkL, or LPS and/or IFN-{gamma}, as indicated, were analyzed for STAT tyrosine 701 phosphorylation by Western blotting with a phosphospecific STAT1 antiserum. C, Macrophages, either untreated or after treatment with hkL for 8 h, were subsequently stimulated with the indicated doses of IFN-{gamma} for 20 min. STAT1 tyrosine 701 phosphorylation was determined by Western blotting with a phosphospecific STAT1 antiserum.

 
Reduced expression of signaling components upstream of STAT1 in Listeria-infected macrophages would provide an explanation for the decreased STAT1 activation in response to IFN-{gamma}. In fact, Mycobacterium avium was reported to diminish expression of the IFN-{gamma}R1 receptor chain (44). In the case of L. monocytogenes, we observed a slight reduction in the expression of IFN-{gamma}R1 on the cell surface, as determined by flow cytometry (Fig. 3GoA). It is not nearly as pronounced as that observed with M. avium and insufficient to explain the much stronger effect on STAT1 tyrosine phosphorylation by IFN-{gamma}. Expression of the JAK1 and JAK2 kinases as analyzed by Western blot was not significantly altered (Fig. 3GoB). This suggests that reduced activity rather than reduced expression of signal transducers might be responsible for the negative effect of Listeria on STAT1 activation.



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  		FIGURE 3. Effect of L. monocytogenes on the expression of IFN-{gamma} receptor and JAKs. A, Macrophages were left untreated (upper panel) or treated with hkL for 8 h (lower panel). The cells were reacted with biotinylated Ab to the IFN-{gamma}R1 chain and subsequently with PE-streptavidin. Ab-labeled cells were analyzed by flow cytometry. Dashed lines, background of cells reacted with PE-streptavidin; solid lines, cells reacted with biotinyl-first Ab and PE-streptavidin. B, The amounts of JAK1 and JAK2 in whole cell lysates were determined by Western blot either directly (JAK1) or after immunoprecipitation (JAK2).

 
To determine whether Listeria-mediated down-regulation of STAT1 activation produces a decrease of STAT1-mediated transcription, luciferase reporter gene assays were performed in Bac1.2F5 macrophages stably transfected with an STAT1-dependent reporter construct. Consistent with our previous observation for IFN-{gamma} and LPS (18), a brief costimulation of the cells with both IFN-{gamma} and Listeria resulted in increased STAT1 transcriptional activity, compared with stimulation by IFN-{gamma} alone. This is the effect expected from increased phosphorylation at S727. By contrast, longer treatment converted the stimulatory effect of Listeria on the IFN-{gamma} response into suppressive activity (Fig. 4Go). S727 phosphorylation by Listeria persists for many hours (Fig. 1Go). Therefore, the reduced stimulatory activity of IFN-{gamma} on transcription seen between 0.5 and 4 h of pretreatment with Listeria is most likely due to the inhibitory effect on IFN-{gamma}-induced STAT1 tyrosine phosphorylation (Fig. 2GoB).



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  		FIGURE 4. Effect of Listeria on the transcriptional response to IFN-{gamma}. Bac1.2F5 macrophages, stably transfected with a STAT-dependent luciferase reporter gene (clone C11), were either treated with hkL alone for the indicated periods, or were pretreated with hkL for the indicated periods, followed by stimulation with IFN-{gamma} for 2 h, and luciferase activity determined in cell extracts. t = 0 corresponds to 2 h of IFN-{gamma} treatment without Listeria pretreatment. Inducibility represents fold induction of IFN-{gamma}/hkL values over values with hkL alone. hkL treatment alone caused no transcriptional increase at 2.5 h and a 2-fold increase over untreated controls after 6 h of stimulation.

 
Effect of intracellular Listeria on the expression of socs3 gene

The inhibition of STAT1 tyrosine phosphorylation prompted us to investigate a possible role of SOCS proteins in Listeria-mediated signaling. Two members of this family, SOCS1/JAK-binding protein/SSI1 and SOCS3/cytokine-inducible Src homology 2 domain-containing protein 3/SSI3, have been associated with inhibition of the IFN-{gamma} response (18, 45, 46, 47). Northern blots with RNA from Bac1.2F5 macrophages infected with L. monocytogenes for various periods demonstrated increased expression of SOCS3 mRNA (Fig. 5GoA). Maximal levels of SOCS3 mRNA were reached after 4 h of Listeria treatment. Interestingly, the induction was only in part resistant to inhibition of protein synthesis by cycloheximide. After short treatment with Listeria (2 h), the induction was fully resistant to cycloheximide. Longer periods of infection with Listeria (4 h) led to a partial dependence on protein synthesis. SOCS3 protein was induced by L. monocytogenes as well as hkL with maximal levels after 6 h (Fig. 5GoB). This result argues for a causal effect of SOCS3 on suppression of IFN-{gamma}-induced signal transduction, in which a clear effect on STAT1 tyrosine phosphorylation is evident after 4 h and a maximal effect after 8–12 h (Fig. 2Go).

L. monocytogenes infection of macrophages causes I-{kappa}B degradation and the secretion of a SOCS3-inducing polypeptide

NF-{kappa}B is a major player in the transcription of antibacterial and proinflammatory genes. Consistent with a role of NF-{kappa}B in regulating the expression of Listeria-induced genes, its inhibitor, I-{kappa}B{alpha}, was rapidly degraded after stimulation of macrophages with heat-killed bacteria (Fig. 6GoA). Thus, NF-{kappa}B is a candidate transcription factor for the rapid and direct stimulation of SOCS3 mRNA expression by L. monocytogenes.



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  		FIGURE 6. I-{kappa}B degradation and secretion of a SOCS3-inducing activity in response to L. monocytogenes. A, Macrophages were stimulated with hkL for the indicated periods, and I-{kappa}B degradation was determined by Western blotting of whole cell lysates. B, Macrophages were infected with wild-type L. monocytogenes for 4 h. Supernatants were cleared by centrifugation and transferred to cultures of previously untreated macrophages for the indicated periods. Extracts of these cells were analyzed for SOCS3 expression by Western blotting. C, Wild-type or STAT1-deficient bone marrow-derived macrophages were stimulated with hKL, or, as a control, with LPS for the indicated periods. SOCS3 expression in whole cell lysates was monitored using a mAb. D, Cells were treated with hkL or LPS for 6 h. Where indicated, 10 µM SB203580 was added 30 min before subsequent treatment. SOCS3 expression was assayed by Western blotting.

 
We tested whether the indirect synthesis of SOCS3, resulting from cycloheximide-sensitive mRNA induction after Listeria infection, was caused by a secreted polypeptide. To this end, we treated previously unstimulated macrophages with supernatants derived from macrophages 4 h after infection with L. monocytogenes, or after treatment with hkL. This experimental protocol caused significant induction of SOCS3 (Fig. 6GoB).

L. monocytogenes was previously shown to induce the synthesis of IL-10 (48) and IL-10 was reported to induce SOCS3 mRNA (49). To investigate whether the secreted, SOCS3-inducing peptide might be IL-10, we infected macrophages with Listeria in the presence of neutralizing Abs to IL-10. This treatment did not decrease SOCS3 mRNA expression (Fig. 5GoA). Type I IFN were previously shown to be important intermediates in the transcription of the inducible NO synthase gene in response to Leishmania major infection (50). Macrophages isolated from STAT1-deficient mice responded to Listeria infection with SOCS3 synthesis at similar amounts and with similar kinetics, thus ruling out a similar role for type I IFN for Listeria infection and the induction of SOCS3 (Fig. 6GoC). In conclusion, neither IL-10 nor type I IFN appears to be involved in the stimulation of the socs3 gene. Therefore, the cause for sensitivity to cycloheximide after extended periods of Listeria infection is still not clear.

Like the phosphorylation of STAT1S727, Listeria-induced SOCS3 expression was also SB203580 sensitive (Fig. 6GoD). SB203580 in this case caused complete inhibition of SOCS3 expression.

Together the data establish SOCS3 as a direct and indirect Listeria target gene, induced by Listeria-responsive transcription factors or mRNA stabilization, and a secreted polypeptide.

Listeria infection inhibits GM-CSF-mediated JAK-STAT signal transduction

SOCS3 is known to inhibit signaling of a broad array of stimuli, such as prolactin, leptin, growth hormone, GM-CSF, and, as confirmed before, IFN-{gamma} (reviewed in Ref. 51). To confirm SOCS3 activity in Listeria-infected cells, STAT5 activation by GM-CSF was tested in Western blot experiments. Bac1.2F5 macrophages were preinfected with Listeria for the indicated periods and subsequently stimulated with GM-CSF for 25 min. Already after 3 h of pretreatment with Listeria, a decrease in STAT5 tyrosine phosphorylation could be observed in response to GM-CSF when compared with STAT5 expression in the control blot (Fig. 7Go). This effect is consistent with results obtained from EMSA experiments (data not shown).



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  		FIGURE 7. The effect of Listeria pretreatment on STAT5 activation by GM-CSF. Macrophages were either left untreated or were pretreated with hkL for the indicated periods before stimulation with GM-CSF for 25 min. Cellular extracts were analyzed for STAT tyrosine 694/699 phosphorylation by Western blotting with a phosphospecific STAT5 antiserum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pivotal role of STAT1 in immune responses, and for the activation of macrophages in particular, is demonstrated in STAT1 knockout mice (52, 53). In this study, we report that Gram-positive bacteria such as L. monocytogenes can influence STAT1 activity in a time-dependent manner. Short time exposure of macrophages to Listeria leads to phosphorylation of STAT1 at S727, which augments its activity, whereas prolonged infection inhibits STAT1 tyrosine phosphorylation via induction of SOCS3 protein.

Together with our recent findings (8, 18), the results presented in this study suggest that the enhancement of STAT1 activity after a brief contact with macrophages and the suppression of activity after chronic exposure are hallmarks of responses to probably all types of bacteria. They also imply that Gram-negative and Gram-positive bacteria, while using different pattern recognition receptors, elicit signaling pathways that converge at the level of STAT1 activation. p38 MAPK is activated and required for the phosphorylation of STAT1 at S727 not only in response to LPS (8), but also to Gram-positive bacteria such as L. monocytogenes.

Viable bacteria are neither required for the induction of STAT1 S727 kinase nor are they necessary for the inhibition of STAT1 tyrosine phosphorylation. Avirulent mutant Listeria that lack the pore-forming toxin LLO, as well as hkL, reproduce the effects of live bacteria by significantly reducing the level of tyrosine 701-phosphorylated STAT1. This indicates that the bacterial component(s) that initiates inhibition of the IFN-{gamma} response is (are) constitutively expressed and not triggered upon infection of the host cell. The bacterial membrane and its interaction with, e.g., the corresponding TLR, appear entirely sufficient to cause the effects on IFN-{gamma} signal transduction.

As in the case of LPS, SOCS3 mRNA and protein induction in response to Listeria is most likely responsible for the inhibition of STAT1 tyrosine phosphorylation. As a member of a family of feedback inhibitors of cytokine signaling pathways, SOCS3 was shown to be induced by a broad array of stimuli (reviewed in Ref. 51). IL-10 is among these stimuli, but was shown in our studies not to be responsible for the partial sensitivity of SOCS3 mRNA expression to cycloheximide after treatment with Listeria for more than 4 h. Moreover, SOCS3 mRNA was induced in absence of STAT1, ruling out a transcriptional response to type I or type II IFN in this particular aspect of L. monocytogenes infection. In contrast, our data clearly imply an unidentified, secreted cytokine in SOCS3 synthesis. In this aspect, Listeria differs from LPS, in which induction is exclusively due to direct effects on the socs3 gene (18).

Likewise, the signals and transcription factors induced by Listeria to cause direct (i.e., cycloheximide-resistant) SOCS3 expression remain to be clarified. The Toll family receptor TLR2 and postreceptor signals (NF-{kappa}B?) are likely candidates. Like STAT1 phosphorylation at S727, the expression of SOCS3 protein in response to Listeria or LPS is also p38 MAPK dependent, confirming the important role of the enzyme during macrophage activation. At present, we do not know whether p38 MAPK is required for SOCS3 transcription, mRNA stability, or protein translation.

Our studies suggest that STAT S727 kinase activation and SOCS3 induction are general aspects of the antimicrobial response of macrophages. These responses can be caused by cell wall components (LPS or heat-killed bacteria) and therefore do not require active participation of the bacteria. Therefore, it seems unlikely that they represent part of a bacterial survival strategy. We speculate that in an initial phase of infection, it is immunologically meaningful to achieve maximal macrophage activation to eliminate invaders with maximal efficiency. Activation of the STAT1 S727 kinase is part of this host cell strategy because it enhances the output of IFN-{gamma}-induced genes. Upon persistent infection with bacteria, the effects of chronic macrophage activation, hence inflammation, may be detrimental to the host organism. Therefore, deactivation mechanisms are implemented, and intracellular SOCS3 activity is part of these. IL-10-induced SOCS3 production is part of its deactivating activity, but our studies show that both LPS and Gram-positive bacteria can stimulate SOCS3 synthesis directly and independently of deactivating cytokines.

While stressing the significance of Listeria-induced SOCS3 for lowering the inflammatory potential of chronically activated macrophages, our data and results from other labs also suggest a general alteration of the macrophage’s cytokine responsiveness by intracellular SOCS3. This is very evident from the inability of SOCS3-expressing cells to mount a proper response to GM-CSF. The full implications of SOCS3 activity for the physiology of macrophages remain to be determined in future studies.


   Acknowledgments
 
We thank Manuela Baccarini and Pavel Kovarik for helpful suggestions and critical reading of the manuscript. We also thank Pascale Cossart (Institute Pasteur) for providing wild-type and mutant Listeria strains and David Levy (New York University School of Medicine) for STAT1-deficient mice.


   Footnotes
 
1 This work was supported by Grant P12946-GEN from the Austrian Research Foundation (FWF) to T.D. Work in the laboratory of J.K. was supported by Grant 301/00/0564 from the Grant Agency of the Czech Republic. Back

2 Address correspondence and reprint requests to Dr. Thomas Decker, Vienna Biocenter, Institute of Microbiology and Genetics, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. Back

3 Abbreviations used in this paper: JAK, Janus kinase; GAS, IFN-{gamma} activation site; hkL, heat-killed cells of Listeria monocytogenes; I-{kappa}B, inhibitory protein that dissociates from NF-{kappa}B; LLO, listeriolysin O; MAPK, mitogen-activated protein kinase; SOCS, suppressor of cytokine signaling; SSI, STAT-induced STAT inhibitor; TLR, Toll-like receptor. Back

Received for publication March 31, 2000. Accepted for publication September 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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