The production of IFN-γ by T cells and the ability of this cytokine to activate the transcription factor STAT1 are implicated in the activation of antimicrobial mechanisms required for resistance to intracellular pathogens. In addition, recent studies have suggested that the ability of STAT1 to inhibit the activation of STAT4 prevents the development of Th1 responses. However, other studies suggest that STAT1 is required to enhance the expression of T-bet, a transcription factor that promotes Th1 responses. To address the role of STAT1 in resistance to T. gondii, Stat1−/− mice were infected with this pathogen, and their response to infection was assessed. Although Stat1−/− mice produced normal serum levels of IL-12 and IFN-γ, these mice were unable to control parasite replication and rapidly succumbed to this infection. Susceptibility to toxoplasmosis was associated with an inability to up-regulate MHC expression on macrophages, defects in NO production, and the inability to up-regulate some of the IFN-inducible GTPase family of proteins, molecules associated with antitoxoplasma activity. Analysis of T cell responses revealed that STAT1 was not required for the development of a Th1 response, but was required for the infection-induced up-regulation of T-bet. Together these studies suggest that during toxoplasmosis the major role of STAT1 is not in the development of protective T cell responses, but, rather, STAT1 is important in the development of antimicrobial effector mechanisms.
The production of IFN-γ by T cells and NK cells represents a major mechanism of resistance to many bacterial, parasitic, and viral infections (1, 2, 3), but questions remain regarding the many effects induced by this cytokine. IFN-γ up-regulates the expression of >500 genes (1), many associated with immunoregulatory responses. For example, IFN-γ has been shown to affect the production of cytokines and to be a potent enhancer of the expression of MHC I and II (4). However, during infection the effects of IFN-γ have been primarily associated with its ability to activate antimicrobial effector responses of macrophages as well as other cell types (1, 5). The ability of IFN-γ to activate macrophages to produce reactive oxygen and nitrogen intermediates is important in resistance to many pathogens (6, 7). In addition, recent studies have shown that IFN-γ can induce the expression of a family of GTPases (inducibly expressed GTPase (IGTP),3 LRG47, GTPI, IRG47, IIGP, T cell-specific GTP (TGTP)) that are implicated in resistance to several intracellular pathogens (8, 9). The function of these IFN-γ induced proteins in host defense is not clear, but previous studies have shown that acute resistance to Toxoplasma gondii is dependent on IGTP (9) and LRG47 (10), whereas IRG47 has a role during chronic infection (10).
Although STAT1 homodimers are necessary to affect many IFN-γ effector functions, there have been reports of STAT1-independent, IFN-γ-dependent pathways that may be important during infection (11, 12, 13). For example, IFN-γ can induce the expression of a number of genes, such as c-myc, c-jun, SOCS3, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and IL-1β in cells lacking STAT1. Nevertheless, despite the ability of IFN-γ to activate certain genes independently of STAT1, little is known about the role of IFN-γ-dependent, STAT1-independent pathways in resistance to infection.
In addition to a role for STAT1 in the regulation of IFN-γ-mediated, antimicrobial effector mechanisms, several studies have suggested contradictory roles for STAT1 in the development of Th1-type responses. It has been proposed that STAT1 is a negative regulator of Th1 responses through its ability to inhibit the IFN-αβ-induced activation of STAT4 associated with the development of Th1 responses (14). This is supported by studies showing that Stat1−/− mice have enhanced IFN-γ responses during infection with lymphocytic choriomeningitis virus (LCMV) (15), although it is not clear whether this occurs in other experimental systems. In contrast, other studies suggest that the activation of STAT1 by IFN-γ is required for the induction of T-bet, which promotes the development of subsequent Th1-type responses (16, 17), but it is unclear whether this mechanism occurs during infection.
The data presented in this study address the role of STAT1 in resistance to the intracellular parasite, Toxoplasma gondii. Resistance to this pathogen is dependent on the production of IL-12 by accessory cells, such as macrophages, which drives CD4+ and CD8+ T cell production of IFN-γ which, in turn, stimulates infected cells to control parasite replication. Previous studies have reported that patients with defects in STAT1 activation are able to control infection with T. gondii (18), and in vitro infection of macrophages with T. gondii results in an inhibition of STAT1 activation (19). Together, these reports suggest that there may be a role for STAT1-independent mechanisms of resistance to this parasite. However, similar to mice lacking IFN-γ, Stat1−/− mice display an increased parasite burden and succumb to acute infection. This increased susceptibility is associated with defects in several IFN-γ-mediated events, such as up-regulation of macrophage expression of MHC classes I and II (MHC I and II), and NO production. Additionally, in the absence of STAT1, the infection-induced expression of the IFN-γ-inducible GTPases, TGTP and IIGP, was not observed, whereas the expression of IGTP, IRG47, and LRG47 was shown to be partially independent of STAT1. Analysis of parasite-specific T cell responses revealed that STAT1 was not required for the development of a Th1 response (as represented by IFN-γ production), and Stat1−/− mice infected with T. gondii had elevated numbers of activated CD4+ T cells, which produce increased levels of IFN-γ compared with wild-type controls. Furthermore, it appears that STAT1-mediated signaling is required for optimal infection-induced expression of T-bet, but despite the reduced levels of T-bet, Stat1−/− T cells are still capable of developing a normal IFN-γ response. Together, these studies suggest that during toxoplasmosis the major role of STAT1 is not in the development of T cell responses, but in the regulation of multiple IFN-γ-mediated, antimicrobial effector functions required for resistance to T. gondii.
Materials and Methods
Mice and parasites
Female Stat1−/− mice and their wild-type controls (SvEv) were purchased from Taconic Laboratories (Germantown, NY). IFN-γR−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and IFN-αβR−/− mice were bred at University of Pennsylvania (Philadelphia, PA) by Dr. L. Buxbaum. Mice were maintained in Thoren caging units (Thoren Caging System, Hazelton, PA) within the laboratory animal research facilities at University of Pennsylvania. Age-matched mice were infected i.p. with 20 cysts of the ME49 strain of T. gondii, isolated from chronically infected CBA mice (The Jackson Laboratory) as previously described (20).
Analysis of IFN-γ-induced GTPase proteins
Bone marrow-derived macrophages were prepared as previously described (21). Briefly, bone marrow cells were plated at 5 × 106 cells/plate in the presence of DMEM/10% FCS and 35% supernatants of L929 cells. Fresh medium was added on day 3, and on day 6 cells were washed and rested 24 h before use on day 7. On day 7 macrophages were stimulated with IFN-γ (100 U/ml) for 24 h before lysis.
Peritoneal exudate cells (PECs) were obtained by peritoneal lavage with 5 ml of PBS. Whole cell lysates of macrophages or PECs were prepared as follows: 5 × 106 cells were resuspended in PBS, then lysed in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 8), 10 mM EDTA, 10 mM NaF,10 mM NaPO4 (pH 7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), with 10 μM aprotinin and leupeptin, and 3 mM Na3VO4. Lysates were analyzed by SDS-PAGE using a 10% (w/v) acrylamide gel. Protein was transferred to nitrocellulose and blocked in 5% nonfat dry milk in TBS/0.1% Tween for 1 h. Either the mAb α-IGTP (1/10,000; BD Biosciences, San Diego, CA) or one of the polyclonal Abs, α-TGTP (1/1,000), α-IRG47, α-IIGP, or α-GTPI (1/250) and α-actin (1/5,000; Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in 0.5% nonfat dry milk in TBS/0.1% Tween and hybridized to the nitrocellulose overnight at 4°C. The membrane was washed multiple times in TBS/0.1% Tween, and the appropriate secondary Ab was added at a dilution of 1/5,000 (either goat anti-mouse IgG-HRP (Pierce, Rockford, IL) or donkey anti-goat IgG-HRP (Santa Cruz Biotechnology)) in 0.5% nonfat dry milk in TBS/0.1% Tween for 1 h at room temperature. The membranes were then washed multiple times, and signal was detected with ECL reagent (Amersham Pharmacia Biotech, Arlington Heights, IL) as indicated by the manufacturer.
Analysis of cytokine production and iNOS activity
The levels of IFN-γ and IL-12 were measured in serum by ELISA 7 days after infection (22). Splenocyte production of nitrite was measured after stimulation of 4 × 105 cells (in triplicate) with soluble Toxoplasma Ag (STAg; 20 μg/ml) for 48 h. STAg was prepared from tachyzoites of the RH strain of T. gondii as previously described (23). Supernatants from these cultures were assayed for nitrite production using the Greiss assay (24).
Detection of T-bet expression
CD4+ T cells were purified from splenocytes of uninfected and infected mice 7 days postinfection by positive selection. α-CD4-FITC Ab (BD PharMingen, San Diego, CA) was incubated with splenocytes for 20 min on ice. α-FITC-labeled magnetic beads were added to the samples, rocking for 20 min at room temperature. Beads were captured with a magnet and washed twice with DMEM. Unbound cells were then positively selected for CD8+ T cells in the same manner. After a final wash, the beads were resuspended in TRIzol reagent (Invitrogen, Carlsbad, CA), and total RNA was extracted according to the manufacturer’s protocol. The samples were equalized for rRNA on ethidium bromide-nondenaturing 1% agarose gels for Northern blot analysis. The equalized RNA samples (5–20 μg) were resolved on a denaturing 1% agarose-formaldehyde gel and transferred overnight to a nitrocellulose membrane. The membrane was cross-linked and prehybridized for 4 h at 42°C in a solution containing 50% formamide, 5× SSC, 1× Denhardt’s solution, 25 mM sodium phosphate (pH 6.5), and 250 μg of Torula RNA/ml. Hybridization was performed overnight under similar conditions, except for the addition of 10% dextran sulfate and 0.8 × 106 cpm of DNA probe/ml solution. To generate murine probes, primer sequences used are as follows: T-bet: sense, 5′-CGG AGC GGA CCA ACA GCA TCG TTT C-3′; and antisense, 5′-CAG GGT AGC CAT CCA CGG GCG GGT A-3′; and actin: sense, 5′-TCA CCC ACA CTG TGC CCA TCT ACG-3′; and antisense, 5′-TCA CCC ACA CTG TGC CCA TCT ACG-3′. DNA probes were labeled with 32P by random priming using the DECAprimeI Random Priming DNA Labeling Kit (Ambion, Austin, TX) and were purified on NICK columns (Amersham Pharmacia Biotech). After hybridization, the membrane was washed briefly at room temperature in 2× SSC/0.1% SDS and then twice for 30 min each time at 46°C in 0.1× SSC/0.1% SDS. Overnight and 5-day exposures were recorded using storage phosphor screens and cassettes (Amersham Pharmacia Biotech). Northern blot mRNA was quantitated using MultiAnalyst software (Bio-Rad, Hercules, CA). The adjusted volume count of the T-bet mRNA was divided by the adjusted volume count of the actin mRNA, and this number was multiplied by 100 to give the values shown in Fig. 8.
Flow cytometric analysis
To assess T cell and macrophage expression of activation markers, splenocytes or PECs were resuspended in FACS buffer (1× PBS, 0.2% BSA, and 4 mM NaN3) and incubated with Fc block for 30 min on ice. Cells were then stained for various surface markers (splenocytes: CD44-PE, CD62L-allophycocyanin, CD4-PerCP, and CD8-FITC; PECs: IA-PE, H-2Kb-FITC, Mac-3-FITC, and Mac3-PE; BD PharMingen) for 20 min on ice. Cells were washed with FACS buffer and resuspended in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in PBS. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed with CellQuest software (BD Biosciences). For intracellular cytokine staining, after 48-h incubation with STAg (20 μg/ml), splenocytes were incubated with brefeldin (10 μg/ml; Sigma-Aldrich) for 3 h and then were surface-stained with CD4-PE and CD8-FITC (BD PharMingen) and fixed in 1% paraformaldehyde. Cells were permeabilized with 0.1% saponin (Sigma-Aldrich) in FACS buffer and stained for intracellular IFN-γ (using IFN-γ-allophycocyanin; BD PharMingen) for 25 min on ice. Cells were washed with saponin, followed by two washes and resuspension in FACS staining buffer before collection. Ab concentrations were empirically determined for optimal staining.
Cytological analysis for parasite burden
Cells were collected at the site of infection by peritoneal lavage, and cytospins were prepared with 5 × 104 cells/100 μl. Slides were stained with Protocol Hema3 stain (Biochemical Sciences, Swedesboro, NJ) as recommended by the manufacturer and were mounted and sealed with Cytoseal (Stephens Scientific, Kalamazoo, MI).
Unpaired two-tailed Student’s t tests were calculated using INSTAT software (GraphPad, San Diego, CA). A value of p < 0.05 was considered significant.
Stat1−/− mice fail to control parasite infection
STAT1 is activated by both IFN-γ and IFN-αβ. To determine the role of STAT1 in resistance to toxoplasmosis as well as to elucidate the effect of loss of signaling through the IFN-γR or IFN-αβR, wild-type, Stat1−/−, IFN-γR−/−, and IFN-αβR−/− mice were infected i.p. with 20 cysts of T. gondii, and survival was assessed. Stat1−/− mice and IFN-γR−/− mice succumbed to infection within 10–12 days, whereas IFN-αβR−/− and wild-type mice survived past the acute phase of infection (Fig. 1,A). Based on these observations, it appears that the type I IFNs play little role during acute toxoplasmosis. Rather, IFN-γ-mediated signaling through STAT1 is central to resistance to this pathogen. Stat1−/− mice had an increased parasite burden compared with wild-type controls (Fig. 1,B), with approximately 50% of the peritoneal cells being infected by day 7 postinfection. PECS from infected wild-type mice had few cells infected with T. gondii (<2%), but the macrophages displayed an activated phenotype, and there were large numbers of lymphocytes, although few neutrophils (Fig. 1,C). Examination of infected Stat1−/− peritoneal cells revealed large numbers of free parasites in addition to infected cells containing multiple parasites as well as a prominent infiltrate of neutrophils (Fig. 1,D). Histological analysis of liver, lung, and spleen of Stat1−/− mice on day 7 postinfection revealed the presence of prominent macrophage and neutrophil infiltrates and low numbers of parasites. In contrast, there were few neutrophils and less inflammation in tissues from wild-type mice, and parasites were not observed. As activated macrophages are responsible for many antimicrobial mechanisms during infection with intracellular pathogens, the role of STAT1 in the activation of infiltrating macrophages at the site of infection was assessed. PECs from uninfected and infected wild-type and knockout mice were stained for the macrophage surface marker, Mac3, and the levels of MHC I and II on these cells were determined. Both wild-type and Stat1−/− Mac3+ cells from uninfected mice express basal levels of MHC I and II. However, after infection, macrophages from wild-type mice expressed high levels of MHC I and II (Fig. 2, A and C), whereas Stat1−/− macrophages show little or no up-regulation of these surface molecules (Fig. 2, B and D). These data are consistent with a requirement for STAT1 in the IFN-γ induced activation of macrophages at the local site of infection. These in vivo observations are in concordance with a previous study that reported that mouse kidney fibroblasts lacking STAT1 failed to up-regulate levels of MHC class I after stimulation with IFN-γ (25).
As the ability to control replication of T. gondii is dependent on the IL-12 induced production of IFN-γ, systemic levels of these cytokines were assessed to determine whether a defect in the production of these cytokines was associated with the failure of Stat1−/− mice to control T. gondii. Analysis of serum 7 days after infection showed that wild-type and Stat1−/− mice produced comparable amounts of IL-12 after infection (Fig. 3,A), and similar levels of IFN-γ could be detected in the serum of both wild-type and knockout infected mice (Fig. 3 B). Although Stat1−/− mice are able to produce normal levels of IFN-γ, IFN-γ cannot activate its signaling pathway, and these mice behave similarly to IFN-γ−/− mice (26), in that they are unable to control parasite replication and succumb to acute toxoplasmosis. Therefore, these data suggest that Stat1−/− mice infected with T. gondii have a major defect in IFN-γ-mediated antimicrobial activity.
IFN-γ effector functions are defective in Stat1−/− mice
The ability of IFN-γ to activate macrophages to produce NO, an effector molecule important in the inhibition of parasite growth, has been shown to be dependent on STAT1 (25, 27). Therefore, to assess the role of STAT1 in regulating NO production during toxoplasmosis, splenocytes from infected mice were cultured in vitro with soluble Toxoplasma Ag (STAg) for 2 days, and nitrite production was measured using the Greiss assay. Although splenocytes from infected wild-type mice produced high levels of nitrite, these levels were markedly reduced in the absence of STAT1 (Fig. 4). This lack of NO production is consistent with a defect in IFN-γ effector functions and may contribute to the increased parasite burden seen in the Stat1−/− mice.
IFN-γ induces up-regulation of the 47-kDa family of GTPases in vitro (28), and in vivo three of these molecules have been shown to play a role in resistance to toxoplasmosis (9, 10), although it is not clear how this family of molecules mediates antimicrobial function. To further assess the role of STAT1 in resistance to T. gondii, studies were performed to examine its role in the regulation of the IFN-γ-inducible GTPases. Whole cell lysates were prepared from spleens of infected and uninfected mice, and Western blots were used to examine the expression of these molecules. When splenocytes or PECS were used, the Abs for IIGP, LRG47, and IRG47 resulted in the presence of multiple bands, which made the blots difficult to interpret. However, when bone marrow-derived macrophages were used, these Abs gave highly specific signals. In contrast, the Abs for IGTP and TGTP gave highly specific bands of the predicted sizes for these proteins whether the samples were from splenocytes or bone marrow-derived macrophages (Fig. 5). Samples from uninfected wild-type mice displayed baseline levels of IGTP and TGTP, whereas only low levels of IGTP could be detected in samples from Stat1−/− mice (Fig. 5,A). In wild-type mice, by day 7 postinfection there was a marked increase in the expression of IGTP and TGTP, but in the absence of STAT1 this was not observed. Analysis of PECS from wild-type and Stat1−/− mice revealed a similar pattern of expression (data not shown). As the samples from infected wild-type and Stat1−/− mice differed markedly in their cellular composition and parasite burden (see Fig. 1, C and D), in vitro studies were performed to determine whether STAT1 was required for the IFN-γ-induced expression of the 47-kDa family of GTPases. Therefore, bone marrow-derived macrophages from wild-type and Stat1−/− mice were stimulated with IFN-γ for 24 h, and whole cell lysates were prepared for Western blot analysis (Fig. 5,B). This analysis revealed that wild-type and Stat1−/− macrophages expressed basal levels of IGTP, LRG47, TGTP, and IRG47, but no IIGP (Fig. 5 B). When wild-type macrophages were stimulated with IFN-γ, the expression of IGTP, IIGP, and TGTP was markedly up-regulated, whereas less remarkable effects were observed with LRG47 and IRG47. In contrast, when Stat1−/− macrophages were stimulated with IFN-γ, there was no up-regulation in the expression of any of these proteins. Together these studies indicate that although basal expression of IGTP, LRG47, TGTP, and IRG47 is independent of STAT1, the ability of IFN-γ to up-regulate IGTP, IIGP, and TGTP is completely dependent on the activation of STAT1. Together these ex vivo and in vitro studies indicate a critical role for STAT1 in the ability of IFN-γ to stimulate antimicrobial effector mechanisms.
T cell activation is not deficient in Stat1−/− mice
IL-12 and IFN-γ are required for the development of the Th1 immune response necessary for the resolution of T. gondii infection. One in vitro model suggests that STAT1 is necessary for the development of a Th1 response (16, 17), although another model suggests that STAT1 is a negative regulator of IFN-γ production (15). Therefore, to examine the role of STAT1 in T cell responses during toxoplasmosis, the expression of the activation markers CD44 and CD62L was assessed on CD4+ and CD8+ T cells from uninfected and infected wild-type and Stat1−/− mice. Infection resulted in a marked increase in the number of CD44highCD62Llow T cells in both wild-type and Stat1−/− mice (Fig. 6). Moreover, although wild-type and Stat1−/− CD8+ cells displayed comparable increases in activation after infection (Fig. 6,B), there was a significant increase in the numbers of activated Stat1−/− CD4+ T cells compared with wild-type CD4+ T cells (p = 0.032; Fig. 6 A). After infection, there was greater expansion of Stat1−/− splenocytes, resulting in ∼1.5× more cells from the Stat1−/− mice. Therefore, the total number of T cells was higher than that in wild-type mice, but the percentages of CD4+ and CD8+ T cells in Stat1−/− mice were similar to the percentages found in wild-type mice.
Intracellular levels of IFN-γ were measured to determine the role of STAT1 in T cell function. Splenocytes from uninfected and infected wild-type and Stat1−/− mice were stimulated for 2 days with STAg and then stained for CD4, CD8, and intracellular IFN-γ. There was no defect in IFN-γ production after infection, and the amounts of intracellular IFN-γ measured were increased in Stat1−/− mice (Fig. 7). Little IFN-γ was measured in T cells from uninfected mice, but after infection the baseline levels of IFN-γ were increased, with Stat1−/− CD4+ T cells displaying a significantly higher baseline of IFN-γ in their CD4+ T cells than the wild-type CD4+ T cells (p < 0.05). Stimulation with parasite Ag led to increased IFN-γ production by all T cells (p < 0.05), with Stat1−/− CD8+ T cells showing the greatest increase in IFN-γ production after stimulation with STAg. Together, these data demonstrate that during toxoplasmosis, STAT1 is not necessary for the activation of T cells or the development of a Th1 response, and indicate that in the absence of STAT1, there are enhanced IFN-γ responses.
T-bet expression is up-regulated after infection
Recent work has identified a critical role for STAT1 in the up-regulation of the transcription factor T-bet, which is required for the development of Th1 responses (16, 17). However, as Stat1−/− mice infected with T. gondii do not have a defect in the development of a Th1 response, studies were performed to determine whether infection with T. gondii led to increased expression of T-bet and whether STAT1 regulated its expression. CD4+ and CD8+ T cells were purified from uninfected and infected wild-type and Stat1−/− mice, and RNA was extracted for Northern blot analysis of T-bet. Consistent with previous studies (29), Northern blots failed to detect T-bet mRNA in CD4+ and CD8+ T cells from uninfected mice (data not shown). In contrast, CD4+ and CD8+ T cells from infected wild-type mice expressed T-bet mRNA, with the highest levels present in the CD4+ T cells (Fig. 8A). However, although T cells from infected Stat1−/− mice do not have a defect in their ability to produce IFN-γ, there was a marked decrease in the levels of T-bet mRNA compared with T cells from wild-type mice (Fig. 8 B). Thus, STAT1-mediated signaling is required for optimal infection-induced expression of T-bet, but the reduced level of T-bet induced in Stat1−/− T cells after infection is sufficient for the development of a normal IFN-γ response.
The data presented in this study have shown that STAT1 is necessary for the IFN-γ-mediated control of the intracellular pathogen, T. gondii. Although mice lacking STAT1 produced high levels of IFN-γ after infection, they were unable to control parasite replication at the site of infection, and this was associated with reduced expression of molecules required for resistance to T. gondii. Specifically, the IFN-γ-mediated production of NO observed in wild-type mice did not occur in the absence of STAT1. Although this pathway is not essential for resistance to acute toxoplasmosis (30), it does have an important role in the ability of macrophages to control T. gondii (31). More relevant to the acute susceptibility of Stat1−/− mice to T. gondii are the defects observed in the expression of IFN-γ-induced GTPase family members in the absence of STAT1. Thus, STAT1 is required for the optimal infection-induced expression of several members of the IFN-γ-inducible GTPases. As previous studies demonstrated that IGTP and LRG47, but not IRG47, are essential for resistance to acute toxoplasmosis (9, 10), the requirement of STAT1 for the increased expression of IGTP and LRG47 provides a likely explanation for the inability of Stat1−/− mice to control this infection. These findings are consistent with recent studies by Collazo and colleagues (32), who demonstrated that Stat1−/− mice are highly susceptible to T. gondii, and that STAT1 is required for the infection-induced up-regulation of IGTP. Whether the reduced expression of other members of this family of GTPases contributes to the increased susceptibility of Stat1−/− mice to T. gondii is unclear, as the roles of IIGP and TGTP in resistance to this pathogen are unknown. Moreover, the function of these IFN-γ-induced proteins in host defense is not defined, although several family members have been shown to localize to the surface of the endoplasmic reticulum, suggesting a role in protein trafficking or processing (33).
Several reports have shown that STAT1-deficient mice are susceptible to a variety of parasitic, bacterial, and viral infections (25, 32, 34, 35). However, the role of STAT1 in resistance to infections in humans is less clear. Recent studies have identified patients with defects in the IFN-γ receptor that results in reduced (but not absent) activation of STAT1, and although these patients display an increased susceptibility to mycobacterial and Salmonella infections, they are not more susceptible to viral infections or T. gondii (18). It appears that in these patients the reduced ability of IFN-γ to activate STAT1 can be compensated for by TNF-α, which allows macrophages to control replication of T. gondii. Similarly, patients with a heterozygous mutation in STAT1 are more susceptible to Mycobacteria spp. infection, but not to viral infection (36). However, patients who have a homozygous mutation, located in the SH2 domain of STAT1, have a more severe phenotype, leading to increased susceptibility to mycobacterial and viral infection (37), although it is not known whether these individuals are more susceptible to T. gondii.
To date, the studies described focus on the role of IFN-γ-induced activation of STAT1 in the regulation of antimicrobial effector mechanisms. However, IFN-γ also has major immunoregulatory effects that were severely compromised in Stat1−/− mice infected with T. gondii, as evidenced by the reduced capacity to up-regulate MHC I and II at the local site of infection. Nevertheless, T cell activation was not defective in Stat1−/− mice. On the contrary, Stat1−/− T cells were hyperactive and produced more IFN-γ than their wild-type counterparts. The basis for these enhanced T cell responses is unclear, but there are several possible explanations that may contribute to these observations. For example, the increased levels of parasite Ag present in Stat1−/− mice may stimulate enhanced T cell responses, although Levy and colleagues (38) previously showed that Stat1−/− T cells are hyperproliferative, and that this effect was independent of the antiproliferative effects of IFN-γ. However, the increased numbers of activated CD4+ T cells and enhanced IFN-γ responses observed in infected Stat1−/− mice are consistent with studies that have suggested that STAT1 is a negative regulator of Th1 responses (15). Regardless of the mechanism, it appears that in at least two models of infection (toxoplasmosis and LCMV) (15), STAT1 may have a role in limiting IFN-γ responses. However, the enhanced T cell responses observed in Stat1−/− mice infected with LCMV or T. gondii are hard to reconcile with other studies that suggested that activation of STAT1 in T cells is necessary to up-regulate T-bet, which is required to mount a Th1 response (16). This latter model is supported by a recent study showing that after infection with Leishmania major, the ability to generate a protective Th1-type response is dependent on STAT1 (35). However, Afkarian et al. (17) showed that although T-bet expression is decreased in Stat1−/− Th1 cells, these levels of T-bet are sufficient for normal Th1 development. Although these models of the role of STAT1 in the development of IFN-γ responses are fundamentally different, it is possible that the conditions under which Th1 differentiation occurs during infection may determine which activity of STAT1 is dominant. Thus, after infection with pathogens such as LCMV or T. gondii, which have systemic effects and are potent inducers of IL-12, the development of a Th1-type response is not dependent on the STAT1-mediated expression of T-bet. Rather, in these circumstances, the lower levels of T-bet are sufficient to allow the development of protective Th1-type responses.
We thank Dr. Laurence Buxbaum for generously providing us with the IFN-αβR−/− mice, and Dr. Gopa Biswas for help with mRNA quantitation.
This work was supported by National Institutes of Health Grants AI42334 and AI42370, and the State of Pennsylvania.
Abbreviations used in this paper: IGTP, inducibly expressed GTPase; TGTP, T cell-specific GTP; PEC, peritoneal exudate cell; LCMV, lymphocytic choriomeningitis virus; STAg, soluble Toxoplasma Ag.