INFECTION AND IMMUNITY, Apr. 2003, p. 2095–2101 0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.4.2095–2101.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 4
Leishmania donovani-Induced Expression of Suppressor of Cytokine Signaling 3 in Human Macrophages: a Novel Mechanism for Intracellular Parasite Suppression of Activation Sylvie Bertholet,1† Harold L. Dickensheets,2 Faruk Sheikh,2 Albert A. Gam,1 Raymond P. Donnelly,2 and Richard T. Kenney1* Division of Bacterial, Parasitic, and Allergenic Products1 and Division of Therapeutic Proteins,2 Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received 30 July 2002/Returned for modification 6 November 2002/Accepted 24 December 2002
Leishmania donovani protozoan parasites, the causative agent of visceral leishmaniasis, establish an infection partly by interfering with cytokine signaling in the host macrophages. Therefore, we investigated the expression of the suppressor of cytokine signaling (SOCS) genes in human macrophages infected with L. donovani. The expression of SOCS3 mRNA was induced transiently after exposure to live or heat-killed parasites, but not purified lipophosphoglycan, while that of other SOCS genes remained unchanged. SOCS3 gene expression was not dependent on phagocytosis or on cytokines released by L. donovani-infected macrophages, such as interleukin-1 or tumor necrosis factor alpha. In addition, Leishmania used a different signaling pathway(s) than bacterial lipopolysaccharide to induce SOCS3 mRNA, as indicated by the kinetics of induction and sensitivity to polymyxin B inhibition. Finally, phosphorylation of the STAT1 transcription factor was significantly reduced in L. donovani-infected macrophages and required de novo transcription. The induction of SOCS3 provides a potent inhibitory mechanism by which intracellular microorganisms may suppress macrophage activation and interfere with the host immune response. Leishmania donovani, a protozoan parasite, is the causative agent of visceral leishmaniasis, a fatal disease if left untreated that threatens millions of people living in or traveling to tropical and subtropical regions where leishmaniasis is endemic. L. donovani has a dimorphic life cycle defined by a promastigote form within the sand fly vector and an amastigote stage present in the mammalian host. Upon infection, L. donovani promastigotes are rapidly phagocytosed by and eventually establish a persistent infection within host macrophages (m) (1). The initial host defense against Leishmania is executed by innate immunity, involving primarily m and to some extent Langerhans cells (4). To survive inside m and escape immunity, Leishmania has developed mechanisms that deactivate m immune functions, including the inhibition of the respiratory burst, inhibition of interleukin-12 (IL-12) and nitric oxide synthesis, and the downregulation of major histocompatibility complex (MHC) class II molecules, as well as promoting the synthesis of inhibitory cytokines like transforming growth factor  (TGF-) and IL-10 (reviewed in reference 5). Cytokines, and especially gamma interferon (IFN-␥), are essential contributors to m activation to promote effective killing of the parasites. The recent identification of the suppressor of cytokine signaling (SOCS/CIS/JAB/SSI) family (11, 18, 26) presents a new potential mechanism for the inhibitory effect of L. donovani on the cytokine activation of m. The expression of SOCS1 and
SOCS3 in m is induced by multiple cytokines and growth factors (reviewed in reference 15), as well as lipopolysaccharide (LPS) (27) and gram-positive bacteria (28). While SOCS1 has been proposed to inhibit Janus kinase (JAK) activity by binding to the kinase activation loop (29), the mechanism of SOCS3 inactivation is still unclear. The association of SOCS3 with JAKs (2, 24) or with the phosphorylated receptor chain (16, 20, 24) has been postulated. In all cases, the signal transduction cascade is interrupted by lack of appropriate phosphorylation of STAT transcription factors, which inhibits the activation of m. Because L. donovani has been previously shown to interfere with cytokine signaling (references 3, 17, and 23 and references therein), it is important to investigate the level of expression of SOCS genes in L. donovani-infected m. We report here that L. donovani promastigotes specifically induce the expression of SOCS3 mRNA in human m, while the expression of other SOCS genes remains unchanged. Possible mechanisms leading to the expression of SOCS3 in L. donovani-infected m, as well as the effect on STAT1 phosphorylation, are also discussed. MATERIALS AND METHODS Parasite and bacterial strains. L. donovani (strains FDA97052 and MHOM/ SD/62/1S-C12D), Leishmania major (strain MHOM/IL/80/Friedlin), and Leishmania amazonensis (strain FDA99028) were maintained as promastigotes at 25°C as described previously (23). Heat-killed L. donovani parasites were prepared by incubating parasite suspensions at 56°C for 10 min. Parasites tested below the detection limits for endotoxin (⬍0.25 endotoxin unit/ml in the Limulus amebocyte lysate assay; Endosafe, Charleston, N.C.). Purified lipophosphoglycan (LPG) was kindly provided by David Sacks and used as indicated. Mycobacterium bovis (provided by Frank Collins), Listeria monocytogenes (provided by Karen Elkins), and fixed Staphylococcus aureus (Cowan 1 strain; Calbiochem, La Jolla, Calif.) were used at a m/bacterium ratio of 1:20, 1:20, and 2:10,000 (or 0.02%), respectively.
* Corresponding author. Present address: Iomai Corporation, 20 Firstfield Rd., Suite 250, Gaithersburg, MD 20878. Phone: (301) 5564521. Fax: (301) 556-4501. E-mail:
[email protected]. † Present address: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. 2095
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Antibodies and reagents. Neutralizing antibodies to human IL-10 and IL-1 (Endogen, Rockford, Ill.), antibody to tumor necrosis factor alpha (TNF-␣), and control isotype mouse immunoglobulin G1 (IgG1) (PharMingen, San Diego, Calif.) were used at 1 g/ml. Rabbit polyclonal antibodies to STAT1 and phospho-STAT1 (Y701) (Cell Signaling Technology, Beverly, Mass.) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, Calif.) were used for standard Western blots. Recombinant human IFN-␥ (rhIFN-␥) (Genzyme, Cambridge, Mass.), IL-1, TNF-␣, and IL-10 (Endogen) were used at 1 ng/ml. Latex beads, polymyxin B, and actinomycin D were obtained from Sigma (St. Louis, Mo.). Culture, differentiation, and infection of human m. CD14⫹ monocytes were purified by elutriation of pheresed cells from healthy normal volunteer blood donors at the NIH Clinical Center Department of Transfusion Medicine. Monocyte-derived m were obtained by culturing monocytes for 7 days at 2 ⫻ 106 cells/ml in RPMI 1640 medium (Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum, 50 g of gentamicin per ml, and 50 ng of m colony-stimulating factor per ml at 37°C in a humidified 5% CO2 atmosphere. m were exposed for 45 min, when not otherwise indicated, to stationary-phase Leishmania promastigotes (m/parasite ratio of 1:20), heat-killed L. donovani (1:20), latex beads (0.005%,) or LPS (10 ng/ml). For the determination of STAT1 phosphorylation, m were infected with L. donovani for the indicated periods and further incubated for 15 min with 1 ng of rhIFN-␥ per ml. In some experiments, m were preincubated for 1 h at 37°C with 1 mg of actinomycin D per ml. The rate of cellular infection was more than 90% after 2 h of infection as determined by microscopic examination of stained preparations. Briefly, 2 ⫻ 105 infected cells were centrifuged onto microscope slides for 5 min at 500 rpm in a cytocentrifuge (Cytospin; Shandon Southern, Pittsburgh, Pa.), fixed in methanol, and stained successively in eosin and May-Gru ¨nwald solutions according to the manufacturer’s protocol (DiffQuick; Dade AG, Du ¨dingen, Switzerland). RPA. Total RNA was isolated from 5 ⫻ 106 to 10 ⫻ 106 m using RNA STAT-60 (Tel-Test, Inc., Friendwood, Tex.) according to the manufacturer’s instructions. RNase protection assay (RPA) (RiboQuant; PharMingen) was performed according to the manufacturer’s protocol. Briefly, equivalent amounts of RNA (5 to 10 g) were hybridized overnight at 56°C with biotinylated human SOCS, human CK-2b, or human CK-3 multiprobe RNA (20 to 100 ng). RNaseprotected probes were size fractionated by electrophoresis in polyacrylamide gels (QuickPoint; Invitrogen, San Diego, Calif.), and transferred onto positively charged nylon membranes (Roche Molecular Biochemicals, Mannheim, Germany). Biotinylated probes were detected using streptavidin peroxidase and enhanced chemiluminescence (North2South; Pierce, Rockford, Ill.). Human SOCS, CK-2b, and CK-3 multiprobe biotinylated RNA was obtained by transcription of multiprobe template sets using biotin RNA labeling mix (Roche Molecular Biochemicals). Cellular extracts and Western blot analysis. After infection, m (4 ⫻ 106 to 5 ⫻ 106) were washed with cold phosphate-buffered saline (PBS) containing 1 mM sodium orthovanadate and lysed in 100 l of NuPAGE sample buffer (Invitrogen). Aliquots (20 l) were analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis in NuPAGE 4 to 12% Bis-Tris gels using 1⫻ morpholinepropanesulfonic acid (MOPS) running buffer followed by transfer to nitrocellulose membranes. Membranes were probed with phosphoSTAT1 antiserum (at a dilution of 1:1,000), stripped in Restore Western blot stripping buffer (Pierce), and reprobed with anti-STAT1 (1:1,000). A horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody was used at 1:4,000 with enhanced chemiluminescence substrate (SuperSignal; Pierce). Flow cytometry (fluorescence-activated cell sorting [FACS]). After infection, m were washed with cold PBS, fixed, and stained at 4°C in PBS buffer containing 10% normal human serum (to block Fc receptors) and saturating concentrations of fluorescein isothiocyanate-conjugated anti-MHC class II antibodies (Caltag, Burlingame, Calif.) or a biotinylated antibody against IFN-␥ receptor ␣ chain (IFN-␥R␣), followed by phycoerythrin-conjugated streptavidin (PharMingen, Becton Dickinson, San Jose, Calif.). IgG2b isotypes were used to control for nonspecific binding. At least 10,000 events were collected and analyzed using a FACScalibur flow cytometer and CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). The results are expressed in arbitrary units as the mean fluorescence index (MFI), where MFI ⫽ (geometric mean of the antibody/geometric mean of the IgG2b isotype).
RESULTS Upon infection by L. donovani, human m transiently express SOCS3 mRNA. The recent discovery that the SOCS1 and SOCS3 genes are involved in the negative regulation of cyto-
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FIG. 1. L. donovani infection induces SOCS3 gene expression in m. m were exposed to stationary-phase L. donovani (Ld) promastigotes for the times indicated above the lanes. Total RNA was analyzed by RPA for expression of SOCS1 to SOCS6 and CIS, as well as L32 and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. For a control, m were not infected with the L. donovani promastigotes (⫺). The positions of protected probes are indicated by the arrowheads.
kine signaling pathways prompted us to investigate their level of expression in L. donovani-infected m. Analysis by RPA of RNA from m infected for different periods with stationaryphase L. donovani promastigotes demonstrates increased SOCS3 mRNA expression (Fig. 1). The maximal level of SOCS3 mRNA was observed after 30 to 45 min of infection. Interestingly, L. donovani selectively induced SOCS3 mRNA expression in m, whereas other members of the SOCS family were apparently not affected up to 48 h postinfection (Fig. 1 and data not shown). L. donovani RNA tested negative for SOCS expression (data not shown), ruling out the possibility of a parasite contaminant. SOCS3 has been associated with the regulation of numerous signal transduction pathways, notably IFN-␥, IL-4, IL-6, IL-10, and IL-12. These cytokines are essential for m immune functions; therefore, blocking their action through SOCS3 expression may explain at least part of the downmodulation of m activation following Leishmania infection. Different Leishmania species, but not latex bead phagocytosis, induce the expression of SOCS3 mRNA in m. As shown in Fig. 2A, m infected with L. major or L. amazonensis also express SOCS3 mRNA, although the level was lower than that for m infected with the two L. donovani strains. To further determine whether SOCS3 gene expression is dependent on phagocytosis or requires active parasites, RNA from m exposed to latex beads or heat-killed L. donovani were analyzed for SOCS3 gene expression. As demonstrated in Fig. 2B, the phagocytosis of latex beads did not induce the expression of SOCS3, whereas SOCS3 mRNA was detected in response to live or heat-killed L. donovani, arguing for an inducing mechanism independent of the condition of the parasite. This latter observation suggests that parasite surface molecules might be involved as a triggering signal. Parasite LPG is the most abundant molecule on the surface of promastigotes; however, as
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FIG. 2. SOCS3 gene is expressed in m infected with different Leishmania species but is independent of phagocytosis. (A) m were exposed for 45 min to L. donovani (L. donovani [Ld] strains FDA97052 and WHO 1S), L. major (Lm), and L. amazonensis (La). SOCS3 RNA expression is shown in arbitrary units (a.u.). (B) m were exposed for 45 min to L. donovani (Ld), heat-killed L. donovani (hkLd), or latex beads (0.005%). (C) m were exposed for 45 min to L. donovani (Ld), heat-killed Ld (hkLd), or purified LPG. In each panel, m were not infected with a Leishmania species (⫺) as a control. Total RNA was analyzed by RPA for SOCS expression.
shown in Fig. 2C, m did not express SOCS3 mRNA in response to stimulation with 10 to 1,000 ng of purified LPG per ml. m exhibit differential SOCS3 gene expression in response to Leishmania or LPS stimulation. Gram-negative bacterial LPS and gram-positive L. monocytogenes were recently reported to induce the expression of SOCS3 in mouse m (27, 28), so it was of interest to compare SOCS3 gene expression in human m treated with either bacterial stimuli or L. donovani. While stimulation with M. bovis, gram-positive L. monocytogenes, fixed S. aureus, and gram-negative LPS resulted in strong and sustained SOCS3 gene expression after 2 h of treatment, L. donovani-infected m failed to show any increase in SOCS3 RNA at this late time point (Fig. 3A), in agreement with results presented in Fig. 1. The level of SOCS1 RNA was slightly increased in m exposed to bacterial stimuli but not affected in response to L. donovani compared to the uninfected control. In contrast, m exposed for 45 min to L. donovani showed the maximal level of SOCS3 RNA (Fig. 3B), whereas LPS treatment demonstrated only a marginal increase at the same time point (compare Fig. 3B with A). Furthermore, L. donovani-induced SOCS3 gene expression was resistant to polymyxin B treatment, whereas LPS-dependent induction was totally blocked by the drug (Fig. 3B). Together, these observations suggest that L. donovani targets other m receptor(s) or pathway(s) than LPS or the other bacteria do, resulting in differences in kinetics of SOCS3 gene expression and inhibitor sensitivity.
Leishmania-induced expression of SOCS3 in m is independent of cytokine activation. m infected by Leishmania have been reported to release cytokines such as TNF-␣, TGF-, IL-1, and IL-10 (reviewed in reference 5). Since most of these cytokines are also capable of stimulating SOCS3 gene expression, it was of interest to study whether it was the parasites or the cytokines produced by infected m that were responsible for the transient expression of SOCS3 mRNA. m exposed to L. donovani for various time periods were analyzed by RPA for cytokine gene expression, including IL-12, IL-10, IL-1␣, IL-1, IL-1R antagonist, IL-6, IL-18, TNF-␣, TNF-, lymphotoxin , IFN-␥, IFN-, TGF-1, TGF-2, and TGF-3. Of all these cytokines, TNF-␣ and IL-1 mRNA were the only ones upregulated during the first hour following infection with L. donovani promastigotes (data not shown). Within 15 min after L. donovani infection, SOCS3 mRNA, but not IL-1 or TNF-␣, were detected (Fig. 4A), indicating that SOCS3 preceded cytokine gene expression. To further demonstrate that TNF-␣ and/or IL-1 was not responsible for the expression of SOCS3 mRNA, m were exposed to parasites in the presence of cytokine-specific neutralizing antibody or an unrelated IgG control. RNA from m infected with L. donovani demonstrated a consistent increase in SOCS3 mRNA, and neutralizing antibodies to IL-1, TNF-␣, or IL-10 were incapable of modulating L. donovani-induced SOCS3 gene expression (Fig. 4B). At the concentration used (1 g/ml), the antibodies neutralized 1 to 10 ng of the corresponding cytokine per ml, as measured by inhibition of SOCS3 gene expression in cytokine-stimulated
FIG. 3. L. donovani and bacteria differentially induce SOCS3 gene expression in m. (A) m were infected for 2 h with L. donovani (Ld), gram-negative bacterial LPS (10 ng/ml), gram-positive L. monocytogenes, M. bovis, or S. aureus. (B) m were incubated for 45 min with L. donovani (Ld), latex beads, or LPS. Where indicated, polymyxin B (10 g/ml) was present (⫹). Total RNA was analyzed by RPA for SOCS expression. In each panel, m were not infected with L. donovani or bacteria (⫺) as a control.
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FIG. 4. L. donovani-induced SOCS3 gene expression in m is independent of cytokine stimulation. (A) m were incubated for 45 min with L. donovani, and total RNA was analyzed by RPA for cytokine and SOCS3 expression. Relative RNA expression (compared to the total) is shown in arbitrary units (a.u.). (B) m were exposed (⫹) for 45 min to L. donovani (Ld) and neutralizing anti-IL-1, anti-TNF-␣, and anti-IL-10, or irrelevant IgG antibodies (10 g/ml) in the presence of polymyxin B (10 g/ml). Total RNA was analyzed by RPA for SOCS expression.
m (data not shown). Together, these results rule out cytokine-induced SOCS3 expression in L. donovani-infected m and support the hypothesis of a direct signaling mechanism coming from the parasite itself. IFN-␥ signal transduction is altered in Leishmania-infected m expressing SOCS3. m immune functions primarily depend upon IFN-␥ activation. IFN-␥ signals through the JAKSTAT enzymatic cascade, resulting in the tyrosine phosphorylation of STAT1, translocation of the transcription factors to the nucleus, and initiation of gene transcription. Both SOCS1 and SOCS3 have been shown to inhibit the IFN-␥ signaling pathway. To address whether the presence of SOCS3 in L. donovani-infected m affected the capability of m to respond to IFN-␥ stimulation, the phosphorylation level of STAT1 was determined by Western blot analysis (Fig. 5A and B). Uninfected m responded to IFN-␥ stimulation by specifically phosphorylating the tyrosine residue of STAT1 protein, as demonstrated by a phospho-specific antiserum to STAT1 (Y701). Analysis of protein extracts from m infected with L. donovani or heat-killed L. donovani, however, showed a time-dependent decrease in STAT1 phosphorylation in response to IFN-␥. Maximal inhibition (40 to 50%) was reached by 60 min postinfection with heat-killed L. donovani (Fig. 5A) and by 90 min with live parasites (Fig. 5B) and gradually returned to normal levels by 6 to 8 h (data not shown). Treating m with actinomycin D prior to infection with L. donovani to prevent transcription and further protein synthesis was enough to restore complete phosphorylation of STAT1 in response to IFN-␥ (Fig. 5B). The expression of MHC class II in response to IFN-␥ stimulation was strongly decreased in L. donovani-infected m, an effect that was maximal (65%) 4 h postinfection (Fig. 5C), confirming that the signal transduction pathway was affected. In addition, exposure of human m to L. donovani parasites resulted in reduced cell surface (Fig. 6) and total cellular content (data not shown) of the IFN-␥R␣. A maximal
decrease of 72% was observed 6 to 8 h following infection (Fig. 6). The effect was transient and returned to normal levels by 24 h. DISCUSSION This study shows a novel association of a eukaryotic microorganism, the protozoan intracellular parasite L. donovani, with the induction of SOCS3 mRNA expression in infected human m. Two other species of Leishmania, L. major and L. amazonensis, which cause cutaneous infections in human hosts, also induced SOCS3 mRNA, although the levels of expression were lower than that in L. donovani-infected m. The list of pathogens shown to induce SOCS3 expression in m now includes L. monocytogenes (28), Leishmania spp., M. bovis, and S. aureus (this study), suggesting a generalized mechanism that initiates the suppression of m activation. Substantial differences were observed in the kinetics of SOCS3 gene expression and inhibition by polymyxin B when L. donovani and bacterial LPS stimuli were compared, suggesting that distinct pathways are used. SOCS3 is part of a negative-feedback loop that regulates cytokine signaling. SOCS3 expression is dependent on STAT transcription factors and can be induced by several cytokines, including IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-11, IL12, IL-13, IFN-␥, TNF-␣ (reviewed in reference 15), and type I interferons (8). Of these cytokines, we observed that the transcription of TNF-␣ and IL-1 genes was indeed induced in L. donovani-infected m, in agreement with previous studies (7), but this followed rather than preceded transcription of the SOCS3 gene. In addition, neither TNF-␣ nor IL-1 was responsible for the expression of SOCS3 mRNA in L. donovaniinfected m, since neutralizing antibodies to these cytokines did not decrease the induction of SOCS3 gene expression in L. donovani-infected m. In addition, IL-10, which is also pro-
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FIG. 5. L. donovani infection inhibits m STAT1 tyrosine phosphorylation in response to IFN-␥ stimulation. (A) m were infected for the indicated time with heat-killed L. donovani (hkLd) parasites before stimulation (⫹) with rhIFN-␥ (1 ng/ml). Cellular extracts were analyzed by Western blotting for STAT1 phosphorylation. The membrane was incubated first with antiserum specific for phosphorylated STAT1 (Y701) (pY-STAT1) and then stripped and reprobed for STAT1 expression in each lane. (B) In a similar experiment using live L. donovani (Ld), m were incubated (⫹) with 1 g of actinomycin D (Act.D) per ml prior to the infection where indicated. In panels A and B, m were not infected with L. donovani and not stimulated with IFN-␥ (⫺) for a control. Autoradiograms were scanned, and bands were quantified using the NIH Image 1.60 software. Inhibition of phosphorylation was expressed as a percentage relative to the uninfected control and corrected to the level of STAT1 in each lane. (C) The induction of m MHC class II surface expression, in response to rhIFN-␥ stimulation, was measured by FACS 24 h poststimulation.
duced by m infected with Leishmania (6) and which uses the JAK/STAT pathway, did not account for the induction of SOCS3 mRNA by L. donovani. On the basis of these observations, it is unlikely that cytokines mediate the induction of SOCS3 expression observed in L. donovani-infected cells. The hypothesis that a surface molecule of the parasite might
FIG. 6. L. donovani infection decreases m IFN-␥R␣ content. m were infected for the indicated time with L. donovani (Ld). m were further fixed, stained with an antibody specific for IFN␥R␣, and analyzed by FACS for surface expression. The mean fluorescence index is shown in arbitrary units (a.u.).
be involved is supported by the observation that heat-killed L. donovani is as effective as the live parasite in activating SOCS3 mRNA expression, extending previous observations obtained with Listeria-infected m (28) to a eukaryotic organism. LPG is a highly abundant molecule present on the surface of Leishmania promastigotes and has been previously associated with m dysfunctions including protein kinase C activity (10) and IL-12 (p40) transcription (22). We reported previously that pretreatment of m with LPG had no inhibitory effect on STAT1 phosphorylation in response to IFN-␥ stimulation (23). We show here that purified LPG does not induce the expression of SOCS3 RNA in human m as well, providing another link between the status of SOCS3 expression and the functionality of the IFN-␥ signaling pathway. The parasite molecule(s) responsible for the transient expression of SOCS3 RNA in human m is yet to be discovered. Compared with live L. donovani, heat-killed parasites induce stronger SOCS3 RNA expression and faster inhibition of STAT1 phosphorylation in response to IFN-␥ stimulation in exposed m. This suggests that additional mechanisms may be involved. The observation reported by Dalpke et al. (9) that CpG DNA also induces SOCS3 expression may be another mechanism. Bacterial components inducing SOCS3 gene expression like CpG DNA and LPS signal through different m Toll-like receptors. Stoiber et al. (28) recently reported that the expression of SOCS3 in L. monocytogenes-infected m was
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not dependent on IL-10 but was dependent on p38 mitogenactivated protein kinase, suggesting a role for Toll-like receptors. We are therefore conducting studies to further characterize the possible role of m Toll-like receptors in signaling for the induction of SOCS3 in L. donovani-infected m. IFN-␥ signaling is essential for m activation and elimination of invading parasites. In previous studies, Ray et al. (23) and others (19) reported reduced STAT1 phosphorylation in response to IFN-␥ in L. donovani-infected human U937 cells and peripheral blood monocytes. This study suggests a role for SOCS3 as a possible regulatory mechanism induced by L. donovani to interfere with IFN-␥ signaling in m. This hypothesis is supported by earlier observations that SOCS3 partially reduced STAT1 phosphorylation and/or reporter gene activity in cell lines expressing normal levels of SOCS3 (28) or overexpressing SOCS3 (25, 27). SOCS3 has been shown to associate with tyrosine-phosphorylated receptor chains, thus inhibiting binding and phosphorylation of STATs (16, 20, 24). Furthermore, the expression of SOCS3 may explain the downregulation of the IFN-␥R␣ reported in m infected by L. donovani (23; this study) and M. avium (13), as SOCS3 can also interact with members of the ubiquitinylation protein family and targets receptors to proteosomal degradation (14, 30). The results presented in this study show that maximal IFN-␥R␣ chain downregulation is achieved 6 to 8 h postinfection, which correlates with the time of peak SOCS3 protein expression reported by others (27, 28) and is associated with the proteasomal targeting of the receptor. SOCS3 can also bind to JAK1 (12) and JAK2 (2, 24), inhibiting the kinase activity, though this last assessment is still debated (21). If this last function is verified, the expression of SOCS3 could also explain the reduced JAK2 phosphorylation observed in L. donovani-infected U937 cells and human monocytes (19, 23). In a study involving mouse m, Blanchette and coworkers demonstrated that L. donovani interferes with IFN-␥ signaling by upregulating phosphatase SHP-1 activity, resulting in reduced phosphorylation of JAK2 (3). Whether SHP-1 plays a similar role in L. donovani-infected human m remains to be determined. Taken together, SOCS3 expression is involved in the regulation of a large number of cytokine signaling pathways. Transient induction of SOCS3 in response to infection may be a normal component of innate immunity; however, Leishmania might have adapted to profit from the initial m unresponsiveness to cytokine activation. During these first few hours, promastigotes begin to transform into amastigotes, which are the stage ultimately responsible for causing infection and disease. The induction of SOCS3 by L. donovani provides a potential new mechanism for the suppression of m activation during the initiation of an intracellular parasitic infection.
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29. Yasukawa, H., H. Misawa, H. Sakamoto, M. Masuhara, A. Sasaki, T. Wakioka, S. Ohtsuka, T. Imaizumi, T. Matsuda, J. N. Ihle, and A. Yoshimura. 1999. The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J. 18:1309–1320. 30. Zhang, J. G., A. Farley, S. E. Nicholson, T. A. Willson, L. M. Zugaro, R. J. Simpson, R. L. Moritz, D. Cary, R. Richardson, G. Hausmann, B. J. Kile, S. B. Kent, W. S. Alexander, D. Metcalf, D. J. Hilton, N. A. Nicola, and M. Baca. 1999. The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc. Natl. Acad. Sci. USA 96:2071–2076.
