CD40 Restrains In Vivo Growth of Toxoplasma gondii Independently of Gamma Interferon
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感染与免疫杂志 2006年第3期
Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
ABSTRACT
CD40-CD154 interaction is pivotal for resistance against numerous pathogens. However, it is not known if this pathway can also enhance in vivo resistance in gamma interferon (IFN-)-deficient hosts. This is an important question because patients and mice with defects in type 1 cytokine response can control a variety of pathogens. While blockade of endogenous CD154 resulted in a remarkable increase in parasite load in IFN-–/– mice infected with Toxoplasma gondii, in vivo administration of a stimulatory anti-CD40 monoclonal antibody markedly reduced parasite load. This latter effect took place even in T-cell-depleted mice and was accompanied by induction of macrophage toxoplasmacidal activity. CD40 stimulation restricted T. gondii replication independently of STAT1, p47 GTPases, and nitric oxide. In vivo CD40 ligation enhanced tumor necrosis factor alpha (TNF-) production by T. gondii-infected macrophages. In addition, CD40 stimulation required the presence of TNF receptor 2 to reduce parasite load in vivo. These results suggest that CD40-CD154 interaction regulates IFN--independent mechanisms of host protection through induction of macrophage antimicrobial activity and modulation of TNF- signaling.
INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan of worldwide distribution. Tachyzoites of T. gondii infect any nucleated cell of the host and disseminate throughout the body during the acute phase of infection. Development of cell-mediated immunity results in control but not eradication of the infection (11). The ensuing chronic phase of infection is characterized by the presence of tissue cysts that persist for the life of the infected host. Gamma interferon (IFN-) is crucial for control of the acute phase of infection and prevention of reactivation of chronic T. gondii infection (16, 37, 39). Indeed, IFN-–/– mice are exquisitely susceptible to T. gondii (33). These animals rapidly succumb after infection with even nonpathogenic strains of the parasite.
Despite the pivotal role of IFN- for control of T. gondii, studies in animals point toward the existence of additional mechanisms of protection. SCID mice infected with an avirulent strain of T. gondii die of toxoplasmosis (17, 23) in spite of in vivo production of remarkable amounts of IFN- (22, 31, 34). In contrast, adoptive transfer of T cells to these mice confers protection against T. gondii (4, 25). Similarly, athymic nude mice infected with the parasite die despite continuous treatment with IFN- (38), while adoptive transfer of T cells protects these animals against T. gondii (28). Moreover, TNFR1/2–/– and CD154–/– mice die of toxoplasmosis in the face of IFN- upregulation (30, 44), and administration of exogenous interleukin 12 (IL-12) to IRF-1–/– mice enhances protection against T. gondii through a mechanism that appears independent of IFN- (26). Taken together, these findings suggest the existence of IFN--independent mechanism of host protection that act in concert with IFN- for optimal control of T. gondii.
Studies of patients with immunodeficiencies as well as animal models of infection with other pathogens have also shed light on the complex nature of host protection. Humans can control T. gondii infection when IFN- signaling is defective. Patients with partial IFN-R1 deficiency do not develop toxoplasmosis despite serological evidence of infection with T. gondii (24). Moreover, patients with congenital deficiencies in IFN-- and IL-12-mediated immune response do not appear to be susceptible to pathogens other than atypical Mycobacteria and Salmonella (29). Indeed, animal studies have confirmed the existence of IFN--independent host protection since a variety of intracellular pathogens can be controlled in IFN-–/– and IFN-R–/– mice (40, 41, 45).
Except for the fact that tumor necrosis factor alpha (TNF-) takes a pivotal role in protection in IFN--deficient hosts (24, 40, 41, 45), little else is known about regulation of IFN--independent mechanism of resistance to intracellular pathogens. CD40-CD154 interaction is a signaling pathway central for control of a wide variety of pathogens including T. gondii (6, 9, 20, 30). CD40 is expressed on a variety of hematopoietic and nonhematopoietic cells while CD154 (CD40 ligand) is expressed primarily on activated CD4+ T cells (19, 43). CD40-CD154 interaction regulates many aspects of cell-mediated immunity, including activation of antigen-presenting cells, priming of CD4+ and CD8+ T cells, and stimulation of IL-12/IFN- production (19, 43). Moreover, CD40 stimulation of macrophages induces toxoplasmacidal activity independently of IFN- (1, 3). We conducted studies to determine if in vivo CD40 signaling influences the growth of T. gondii in IFN-–/– mice.
MATERIALS AND METHODS
Mice. Specific-pathogen-free female BALB/c, C57BL/6 (B6), IFN-–/– (BALB/c background), STAT1–/– (129Sv/Ev), and TNF receptor 2–/– (TNFR2–/–; B6 background) were purchased from Jackson Laboratories (Bar Harbor, ME) or Taconic (Germantown, NY). LRG-47–/–, IGTP–/–, IRG-47–/– (8, 42), and wild-type controls (C57BL/6 x 129SvImJ), kindly made available by Gregory Taylor, were also used as sources of macrophages. Animals were 8 to 10 weeks old when used. Each experimental group consisted of 5 to 6 mice. Studies were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati College of Medicine.
Parasites. Tachyzoites of the temperature-sensitive mutant ts4 strain of T. gondii were maintained by infecting human foreskin fibroblasts at 33°C in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum (HyClone, Logan, UT). Cysts of the ME49 strain of T. gondii were obtained from the brains of B6 mice 1 month after intraperitoneal (i.p.) injection of 20 cysts. For experimental infections, mice received i.p. either 20 cysts of the ME49 strain or the indicated number of tachyzoites of the ts4 strain in 0.2 ml of phosphate-buffered saline (PBS). Tachyzoites of a transgenic T. gondii strain that express DsRed in their cytoplasm (T. gondii-DsRed; gift from Boris Striepen) were grown in human foreskin fibroblasts at 37°C. Tachyzoites of the RH strain of T. gondii were maintained as described previously (35).
Antibody treatments. To examine the effects of endogenous CD154, mice were treated with a blocking anti-CD154 monoclonal antibody (MAb) (MR1, 0.2 mg i.p. daily beginning on the day of infection; Taconic) or control hamster immunoglobulin G (IgG; Rockland, Gilbertsville, PA). To stimulate CD40 signaling, mice received protein G-purified anti-CD40 MAb (1C10, 25 μg i.p. daily beginning 1 day prior to infection). A rat IgG2 MAb (GL117) was used as a control. IFN- was neutralized by administration of XMG1.2 (0.5 mg i.p. on days –1 and 3 postinfection). Depletion of T cells was accomplished by injection of anti-CD4 (GK1.5) and anti-CD8 (2.43) MAb (1 mg i.p. of each MAb) 2 days before and the day of infection. Administration of these MAbs causes a 90% reduction of T cells as assessed by flow cytometry.
Peritoneal cell preparations. Peritoneal cells were collected by lavage with 2 ml of ice-cold PBS. Smears of 5 x 104 peritoneal cells per mouse were obtained using a cytocentrifuge (Cytospin; Shandon, Pittsburgh, PA). Slides were fixed and stained with Diff-Quick (Dade Diagnostics, Aguada, Puerto Rico). Five hundred cells were analyzed per sample to determine the percentages of T. gondii-infected cells, the number of tachyzoites per 100 peritoneal cells, and the phenotypic composition of cell preparations. The total number of tachyzoites per peritoneal cavity was calculated using the number of parasites per 100 peritoneal cells and the number of peritoneal cells obtained per mouse.
Macrophages and in vitro infection with T. gondii. Peritoneal macrophages obtained as described previously (1) were cultured on eight-chamber tissue culture glass slides (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) at 5 x 105 cells/ml in complete medium consisting of Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Nonadherent cells were removed after 6 h. Tachyzoites of the RH strain of T. gondii were used to infect monolayers at a ratio of 1.5 to 3 parasites per macrophage. Parasite replication was assessed by light microscopy (3). Bone marrow-derived macrophages were obtained as described previously (1).
Induction of CD154 expression. Purified CD4+ T cells were obtained from IFN-–/– mice 7 days after i.p. infection with 1 x 103 tachyzoites of the ts4 strain of T. gondii. Uninfected IFN-–/– mice were used as controls. Spleen cells from these animals were incubated with anti-CD4-coated magnetic beads (Miltenyi Biotec, Auburn, CA), and rosetting cells were removed with a magnet. Populations of CD4+ T cells were >95% CD4+. CD4+ T cells (5 x 105/ml) and autologous bone marrow-derived macrophages (1.25 x 105/ml) were cultured in 96-well plates containing complete medium. For stimulation with T. gondii, CD4+ T cells were incubated with macrophages challenged with T. gondii tachyzoites at a multiplicity of infection of 4 tachyzoites per macrophage as previously described (36). CD154 induction was assessed at 18 h (predetermined optimal time point for detection of CD154).
Flow cytometry. T cells were incubated with Fc block reagent (BD Biosciences, San Jose, CA), followed by staining with anti-CD3 peridinin chlorophyll protein, anti-CD4 fluorescein isothiocyanate (FITC), and anti-CD154 phycoerythrin and appropriate isotype control MAbs (BD Biosciences). Cells were fixed with 1% paraformaldehyde and were analyzed using a FACSCalibur (BD Biosciences). Expression of CD154 was analyzed on 2 x 104 CD4+ T cells.
For detection of intracellular TNF-, peritoneal cells were incubated in complete medium with or without T. gondii-DsRed (1 tachyzoite per peritoneal cell) or with IFN- (100 U/ml; Peprotech, Rocky Hill, NJ) plus lipopolysaccharide (LPS) (100 ng/ml; Sigma Chemical, St. Louis, MO). Cells were incubated in the presence of brefeldin A (10 μg/ml; Sigma Chemical) for 6 h, followed by staining with either F4/80-FITC (Serotec, Raleigh, NC) or appropriate isotype control MAb. Cells were fixed and permeabilized by using IntraPrep permeabilization reagent (Coulter-Immunotech, Hialeah, FL) according to the manufacturer's instructions. Thereafter, cells were stained with anti-TNF--allophycocyanin or appropriate isotype control MAbs (BD Biosciences). After fixation with 1% paraformaldehyde, cells were analyzed using a FACSCalibur. Forward and side scatter properties as well as F4/80 expression were used to identify macrophages. Expression of DsRed was used to identify infected cells. Expression of intracellular TNF- was analyzed in infected and uninfected macrophages.
Immunofluorescence. To determine whether cell-associated T. gondii-DsRed was intracellular, peritoneal cells incubated with the parasite were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were incubated with blocking buffer without prior permeabilization, followed by incubation with anti-SAG1 MAb (DG52; gift from John Boothroyd). Cells were stained with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Eugene, OR) and examined using a Zeiss Axioplan equipped for epifluorescence microscopy. Intracellular tachyzoites display only red fluorescence, while extracellular ones exhibit both red and green fluorescence.
Cytokine assays. Blood was incubated at room temperature for 1 h, followed by incubation at 4°C for 1 h to induce clot retraction. Samples were spun at 140 x g for 10 min at 4°C. Serum was collected and centrifuged at 1,500 x g for 15 min at 4°C. Concentrations of IFN- in sera were determined by enzyme-linked immunosorbent assay (Endogen, Rockford, IL).
Measurement of nitrite production. Culture supernatants of macrophages were collected 24 h after incubation with stimulatory anti-CD40 or control MAb or IFN- plus LPS and 24 h after addition of T. gondii. The amount of nitrite released was calculated colorimetrically using the Griess reaction (Sigma Chemical). Data are expressed as μM nitrite.
Statistical analysis. Statistical significance was assessed by Student's t test or analysis of variance using InStat, version 3.0 (GraphPad, San Diego, CA).
RESULTS
Blockade of endogenous CD154 increases parasite load in IFN--deficient mice infected with T. gondii. We determined if endogenous CD154 plays a role in restricting the growth of T. gondii in IFN-–/– mice. These mice were infected with 1 x 103 tachyzoites of the avirulent temperature-sensitive mutant ts4 strain of T. gondii, followed by administration of control or blocking anti-CD154 MAb. As shown in Fig. 1, blockade of CD154 caused an increase in the percentage of infected peritoneal cells, the number of tachyzoites per 100 peritoneal cells, and the total number of tachyzoites per peritoneal cavity in IFN-–/– mice ([2.6 ± 0.04]-, [2.86 ± 0.17]-, and [4.3 ± 0.6]-fold increases, respectively; P < 0.02). Thus, endogenous CD40-CD154 interaction restrains in vivo growth of T. gondii independently of IFN-.
The results presented above suggested the presence of CD154 during in vivo T. gondii infection. While CD154 can be expressed by a variety of cells, activated CD4+ T cells are considered a major source of this molecule. Thus, we determined whether T. gondii infection induces expression of CD154 on CD4+ cells. Purified CD4+ T cells obtained from either uninfected or T. gondii-infected IFN-–/– mice were incubated with uninfected or T. gondii-infected autologous bone marrow-derived macrophages. As shown in Fig. 2, CD4+ T cells from T. gondii-infected IFN-–/– mice upregulated CD154 when exposed to parasite-infected macrophages (P < 0.001). Thus, T. gondii infection induces a subpopulation of CD4+ T cells that acquires expression of CD154 in response to the parasite.
In vivo CD40 stimulation decreases parasite load in T. gondii-infected IFN-–/– mice and induces STAT1-independent macrophage anti-T. gondii activity. To confirm the role of CD40 during T. gondii infection, we examined the effects of in vivo administration of a stimulatory anti-CD40 MAb. IFN-–/– mice were infected i.p. with tachyzoites of the ts4 strain of T. gondii and were treated with either stimulatory anti-CD40 or control MAb. As shown in Fig. 3, CD40 stimulation caused a marked decrease in the percentage of infected peritoneal cells, the number of tachyzoites per 100 peritoneal cells, and the total number of tachyzoites per peritoneal cavity (52.3% ± 3.1%, 72.4% ± 5.1%, and 77.6% ± 4.0% inhibition, respectively; P < 0.02; n = 3). The effects of anti-CD40 MAb were similar in mice infected with tachyzoites of the ts4 strain of T. gondii or tissue cysts of the ME49 strain of the parasite (data not shown).
Next, we began to explore the mechanism(s) by which in vivo CD40 ligation reduces parasite load in T. gondii-infected mice. CD40 ligation on antigen-presenting cells can modulate T-cell function (19). Thus, we examined whether T cells mediate reduction in parasite load caused by in vivo administration of anti-CD40 MAb. Depletion of T cells did not alter reduction in the percentage of infected peritoneal cells, the number of tachyzoites per 100 peritoneal cells, and the total number of tachyzoites per peritoneal cavity caused by CD40 stimulation (60.1% ± 4.6%, 69.5% ± 5.0%, and 81.0% ± 7.1% reduction, respectively; P < 0.03; n = 3) (Fig. 3). Thus, a stimulatory anti-CD40 MAb reduced the load of T. gondii in the absence of IFN-. In addition, events that follow CD40 engagement do not need T cells to restrict T. gondii growth.
Macrophages are a major component of the immune response against T. gondii. Therefore, we determined whether in vivo CD40 ligation results in induction of macrophage anti-T. gondii activity independently of IFN-. IFN-–/– mice received either a stimulatory anti-CD40 or control MAb i.p. Peritoneal cells were collected 24 h post-MAb administration. No significant difference in the percentage of large mononuclear cells was noted between samples obtained from mice treated with control or anti-CD40 MAb (control, 87.2% ± 3.7%; anti-CD40, 81.8% ± 1.2%; P = 0.17; n = 3). Macrophages were challenged with tachyzoites of the RH strain of T. gondii and were examined by light microscopy. While macrophages from mice that received control MAb did not exhibit anti-T. gondii activity, macrophages obtained from IFN-–/– mice treated with stimulatory anti-CD40 MAb showed a marked decrease in the infection rate from 1 h to 18 h postchallenge (58.84% ± 2.53% reduction; P = 0.0002; n = 3), indicative of killing of the parasite. In addition, macrophages from mice treated with anti-CD40 MAb had a significantly lower parasite load than control macrophages at 18 h postchallenge (65.6% ± 3.0% reduction; P < 0.0001) (Fig. 4). CD40 stimulation induced anti-T. gondii activity even in monolayers with high percentages of infected macrophages. Monolayers with a 68% infection rate at 1 h postchallenge exhibited a 52.7% ± 2.6% reduction in infection rate at 18 h. Administration of higher amounts of anti-CD40 MAb did not further increase anti-T. gondii activity of macrophages (data not shown). Thus, in vivo CD40 stimulation induces macrophage toxoplasmacidal activity independently of IFN-.
Signal transducer and activator of transcription 1 (STAT1) is a signaling molecule central for induction of macrophage antimicrobial activity and resistance against T. gondii (7, 15, 27). STAT1 is required for induction of NOS2 and p47 GTPases (LRG-47 and IGTP) in macrophages (7, 15, 18), effector molecules that control macrophage antimicrobial activity and mediate resistance against T. gondii (8, 32, 42). Thus, we determined whether in vivo CD40 ligation requires STAT1 to restrict T. gondii growth. STAT1–/– mice infected with tachyzoites of the ts4 strain of T. gondii were treated with either control or stimulatory anti-CD40 MAb. As shown in Fig. 5, CD40 stimulation induced a marked reduction (69.7 ± 4.6%; n = 3; P = 0.01) in parasite load in STAT1–/– mice. In agreement with the restriction of T. gondii growth induced by CD40 in STAT1–/– mice, production of reactive nitrogen intermediates, LRG-47, and IGTP did not mediate anti-T. gondii activity. Peritoneal macrophages obtained from IFN-–/– mice treated in vivo with control or anti-CD40 MAb did not produce nitrite when cultured in vitro with or without T. gondii (<5 μM; n = 3). In contrast, macrophages from both groups of mice produced nitrite in response to in vitro culture with IFN-/LPS (24.9 ± 1.3 mM; n = 3). Finally, CD40 stimulation of bone marrow macrophages from LRG-47–/–, IGTP–/–, IRG-47–/–, and wild-type mice resulted in a similar reduction in parasite load (wild type, 32.9% ± 1.5%; LRG-47–/–, 35.7% ± 4.0%; IGTP–/–, 38.8% ± 4.6%; IRG-47–/–, 39.9% ± 1.6%; P = 0.2; n = 3). Taken together, STAT1, reactive nitrogen intermediates, LRG-47, IGTP, and IRG-47 are not required for CD40 stimulation to restrict T. gondii growth in macrophages.
CD40 ligation modulates TNF- production. In vitro studies revealed that autocrine TNF- is required for induction of toxoplasmacidal activity following CD40 stimulation (1). We examined the effect of in vivo CD40 ligation on TNF- production using flow cytometry and assessment of intracellular levels of this cytokine. Preliminary studies revealed that cells sorted from a macrophage gate as determined by forward and side scatter properties were >93% large mononuclear cells with <4% granulocytes. Next, we used a combination of forward and side scatter properties plus expression of the surface marker F4/80 to study TNF- production. Peritoneal cells from mice treated in vivo with control or anti-CD40 MAb were infected with transgenic T. gondii that express DsRed in the cytoplasm. Staining with antibody against the parasite antigen SAG-1 plus Alexa Fluor 488-conjugated secondary antibody of nonpermeabilized peritoneal cells followed by examination using an epifluorescence microscope revealed that >82% of cell-associated T. gondii was intracellular. Thus, expression of DsRed by flow cytometry was used to identify infected cells. Infection with T. gondii did not affect the expression of intracellular TNF- in F4/80+ macrophages from mice treated with control MAb (Fig. 6) (P = 0.6; n = 3). In contrast, in vivo CD40 ligation resulted in increased production of TNF- in T. gondii-infected F4/80+ macrophages (Fig. 6) (P = 0.01; n = 3). Thus, in vivo CD40 stimulation enhances TNF- production in T. gondii-infected peritoneal macrophages.
TNFR2 is required for CD40-induced reduction of parasite load in IFN--depleted mice. CD40 synergizes with TNFR2 but not TNFR1 to induce anti-T. gondii activity in macrophages (2). We determined if in vivo reduction in parasite load triggered by CD40 stimulation requires the presence of TNFR2. B6 or TNFR2–/– mice were treated with a neutralizing anti-IFN- MAb, followed by infection with tachyzoites of the ts4 strain of T. gondii. No detectable levels of IFN- were noted in sera from T. gondii-infected mice that received anti-IFN- MAb. In addition, while mice treated with a control MAb did not exhibit detectable tachyzoites in peritoneal cavities, parasites were readily noted in IFN--depleted mice (Fig. 7). IFN--depleted B6 and TNFR2–/– mice infected with T. gondii were treated with either stimulatory anti-CD40 or control MAbs. While the stimulatory anti-CD40 MAb caused a marked decrease (81.7 ± 6.4%; P = 0.01) in parasite load in IFN--depleted B6 mice, this effect was not noted in IFN--depleted TNFR2–/– mice (P = 0.8). Thus, TNFR2 is required for CD40 stimulation to reduce the load of T. gondii in IFN--depleted hosts.
DISCUSSION
The immune response against pathogens such as T. gondii is multifaceted. While IFN- is central for control of the parasite, many lines of evidence strongly suggest that there are additional mechanisms of control of T. gondii. Despite the fact that IFN-–/– mice are extremely susceptible to T. gondii, even when infected with a nonvirulent strain of the parasite, we report that the CD40-CD154 pathway restrains parasite proliferation in these mice. In vivo CD40 stimulation decreases parasite load in these mice, while blockade of endogenous CD40-CD154 interaction markedly increases parasite load. These findings are important because they may identify an alternate arm of cell-mediated immunity that acts in concert with IFN- for optimal control of T. gondii, explaining why IFN- appears to be insufficient for control of infection in CD154–/– and TNFR1/2–/– mice (30, 44). Given that the interaction between CD40-CD154 is crucial for resistance against numerous other pathogens, these findings raise the possibility that the CD40-CD154 pathway controls IFN--independent host protection against other pathogens besides T. gondii.
While the CD40-CD154 pathway regulated parasite load in T. gondii-infected IFN-–/– mice, we did not observe a consistent effect of this pathway on survival. These findings are compatible with the fact that mice are extremely susceptible to T. gondii in the absence of IFN- and with the notion that the CD40-CD154 pathway may act in concert with IFN- to enhance resistance against the parasite (3, 30). In apparent contrast to mice, humans with congenital defects in IFN- signaling are not susceptible to toxoplasmosis (24). The findings presented here are of importance because they may explain why these individuals control T. gondii infection.
CD40 signaling controls numerous aspects of cell-mediated immunity that include stimulation of type-1 cytokine response, T-cell priming, and modulation of macrophage antimicrobial activity (19). These effects are likely instrumental in mediating resistance to intracellular pathogens. Our results indicate that CD40 stimulation can restrain in vivo growth of T. gondii independently of IFN- and T cells. Induction of toxoplasmacidal activity in macrophages accompanies in vivo reduction in parasite load induced by CD40 stimulation. This finding suggests that modulation of macrophage function contributes to host protection against intracellular pathogens triggered by CD40.
STAT1 is a key regulator of NOS2 and p47 GTPases (7, 15, 18), effector molecules that mediate control of intracellular pathogens (8, 42). In addition to NOS2 (1), we report that CD40 ligation does not require the presence of STAT1 and the p47 GTPases LRG-47, IGTP, and IRG-47 to restrict the growth of T. gondii. These findings support the contention that CD40 modulates macrophage antimicrobial activity not only independently of IFN- but also independently of a signaling molecule and effector molecules classically controlled by this cytokine.
While TNF- is crucial for resistance against T. gondii during the chronic phase of infection, its role is dispensable during the acute phase of infection (10, 44). In contrast, we find that TNF- signaling is necessary for CD40 stimulation to restrict parasite expansion in acutely infected IFN-–/– mice. These findings are compatible with the evidence that TNF- signaling takes center stage in host protection when IFN--dependent mechanisms of resistance are deficient (40, 41, 45). In addition, this report provides an in vivo correlate to our in vitro studies where we report that CD40 synergizes with TNFR2 to induce macrophage anti-T. gondii activity (2).
T. gondii is a poor inducer of TNF- production by macrophages (3, 5). Moreover, T. gondii can actively block TNF- production by macrophages through manipulation of proinflammatory signaling cascades (12). However, the observation that T. gondii infection induces TNF- production in vivo and the role of macrophages as a source of this cytokine suggest that there are immune events that take place during T. gondii infection that upregulate macrophage production of TNF-. We report that in vivo CD40 ligation increases TNF- production by T. gondii-infected macrophages. The increase in production of this cytokine by macrophages is likely sufficient to trigger toxoplasmacidal activity in macrophages, since low concentrations of TNF- fully synergize with CD40 to induce this effector response (2). Taken together, these findings suggest that CD40 is an important modulator of TNF- signaling during T. gondii infection.
Activated CD4+ T cells trigger anti-T. gondii activity in macrophages through CD40-CD154 interaction (1). The demonstration that T. gondii infection induces parasite-reactive T cells that acquire CD154 expression provides a source of CD154 for CD40-dependent restriction of T. gondii growth. However, while activated CD4+ T cells are considered a major source of CD154, other cells (eosinophils, basophils, mast cells, and platelets) can also express functional CD154 (13, 14, 21). The fact that blockade of endogenous CD154 during the early phase of infection with T. gondii remarkably enhances parasite load raises the possibility that other cells in addition to CD4+ T cells may also activate CD40-dependent mechanisms of control of parasite replication.
The CD40-CD154 pathway is an important regulator of type 1 cytokine response explaining in part why defects in this pathway lead to susceptibility to opportunistic infections. Using a murine model of T. gondii infection, we report that the CD40-CD154 pathway restrains the growth of an intracellular pathogen in IFN--deficient hosts. These findings provide evidence of an unappreciated role of CD40-CD154 signaling in the in vivo immune response to intracellular pathogens.
ACKNOWLEDGMENTS
We thank Gregory Taylor for providing us with macrophages from p47 GTPase-deficient mice and John Boothroyd for making anti-SAG1 MAb available for these studies.
This work was supported by National Institutes of Health grant AI48406 and the American Heart Association, Ohio Valley Affiliate (C.S.S.).
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ABSTRACT
CD40-CD154 interaction is pivotal for resistance against numerous pathogens. However, it is not known if this pathway can also enhance in vivo resistance in gamma interferon (IFN-)-deficient hosts. This is an important question because patients and mice with defects in type 1 cytokine response can control a variety of pathogens. While blockade of endogenous CD154 resulted in a remarkable increase in parasite load in IFN-–/– mice infected with Toxoplasma gondii, in vivo administration of a stimulatory anti-CD40 monoclonal antibody markedly reduced parasite load. This latter effect took place even in T-cell-depleted mice and was accompanied by induction of macrophage toxoplasmacidal activity. CD40 stimulation restricted T. gondii replication independently of STAT1, p47 GTPases, and nitric oxide. In vivo CD40 ligation enhanced tumor necrosis factor alpha (TNF-) production by T. gondii-infected macrophages. In addition, CD40 stimulation required the presence of TNF receptor 2 to reduce parasite load in vivo. These results suggest that CD40-CD154 interaction regulates IFN--independent mechanisms of host protection through induction of macrophage antimicrobial activity and modulation of TNF- signaling.
INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan of worldwide distribution. Tachyzoites of T. gondii infect any nucleated cell of the host and disseminate throughout the body during the acute phase of infection. Development of cell-mediated immunity results in control but not eradication of the infection (11). The ensuing chronic phase of infection is characterized by the presence of tissue cysts that persist for the life of the infected host. Gamma interferon (IFN-) is crucial for control of the acute phase of infection and prevention of reactivation of chronic T. gondii infection (16, 37, 39). Indeed, IFN-–/– mice are exquisitely susceptible to T. gondii (33). These animals rapidly succumb after infection with even nonpathogenic strains of the parasite.
Despite the pivotal role of IFN- for control of T. gondii, studies in animals point toward the existence of additional mechanisms of protection. SCID mice infected with an avirulent strain of T. gondii die of toxoplasmosis (17, 23) in spite of in vivo production of remarkable amounts of IFN- (22, 31, 34). In contrast, adoptive transfer of T cells to these mice confers protection against T. gondii (4, 25). Similarly, athymic nude mice infected with the parasite die despite continuous treatment with IFN- (38), while adoptive transfer of T cells protects these animals against T. gondii (28). Moreover, TNFR1/2–/– and CD154–/– mice die of toxoplasmosis in the face of IFN- upregulation (30, 44), and administration of exogenous interleukin 12 (IL-12) to IRF-1–/– mice enhances protection against T. gondii through a mechanism that appears independent of IFN- (26). Taken together, these findings suggest the existence of IFN--independent mechanism of host protection that act in concert with IFN- for optimal control of T. gondii.
Studies of patients with immunodeficiencies as well as animal models of infection with other pathogens have also shed light on the complex nature of host protection. Humans can control T. gondii infection when IFN- signaling is defective. Patients with partial IFN-R1 deficiency do not develop toxoplasmosis despite serological evidence of infection with T. gondii (24). Moreover, patients with congenital deficiencies in IFN-- and IL-12-mediated immune response do not appear to be susceptible to pathogens other than atypical Mycobacteria and Salmonella (29). Indeed, animal studies have confirmed the existence of IFN--independent host protection since a variety of intracellular pathogens can be controlled in IFN-–/– and IFN-R–/– mice (40, 41, 45).
Except for the fact that tumor necrosis factor alpha (TNF-) takes a pivotal role in protection in IFN--deficient hosts (24, 40, 41, 45), little else is known about regulation of IFN--independent mechanism of resistance to intracellular pathogens. CD40-CD154 interaction is a signaling pathway central for control of a wide variety of pathogens including T. gondii (6, 9, 20, 30). CD40 is expressed on a variety of hematopoietic and nonhematopoietic cells while CD154 (CD40 ligand) is expressed primarily on activated CD4+ T cells (19, 43). CD40-CD154 interaction regulates many aspects of cell-mediated immunity, including activation of antigen-presenting cells, priming of CD4+ and CD8+ T cells, and stimulation of IL-12/IFN- production (19, 43). Moreover, CD40 stimulation of macrophages induces toxoplasmacidal activity independently of IFN- (1, 3). We conducted studies to determine if in vivo CD40 signaling influences the growth of T. gondii in IFN-–/– mice.
MATERIALS AND METHODS
Mice. Specific-pathogen-free female BALB/c, C57BL/6 (B6), IFN-–/– (BALB/c background), STAT1–/– (129Sv/Ev), and TNF receptor 2–/– (TNFR2–/–; B6 background) were purchased from Jackson Laboratories (Bar Harbor, ME) or Taconic (Germantown, NY). LRG-47–/–, IGTP–/–, IRG-47–/– (8, 42), and wild-type controls (C57BL/6 x 129SvImJ), kindly made available by Gregory Taylor, were also used as sources of macrophages. Animals were 8 to 10 weeks old when used. Each experimental group consisted of 5 to 6 mice. Studies were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati College of Medicine.
Parasites. Tachyzoites of the temperature-sensitive mutant ts4 strain of T. gondii were maintained by infecting human foreskin fibroblasts at 33°C in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum (HyClone, Logan, UT). Cysts of the ME49 strain of T. gondii were obtained from the brains of B6 mice 1 month after intraperitoneal (i.p.) injection of 20 cysts. For experimental infections, mice received i.p. either 20 cysts of the ME49 strain or the indicated number of tachyzoites of the ts4 strain in 0.2 ml of phosphate-buffered saline (PBS). Tachyzoites of a transgenic T. gondii strain that express DsRed in their cytoplasm (T. gondii-DsRed; gift from Boris Striepen) were grown in human foreskin fibroblasts at 37°C. Tachyzoites of the RH strain of T. gondii were maintained as described previously (35).
Antibody treatments. To examine the effects of endogenous CD154, mice were treated with a blocking anti-CD154 monoclonal antibody (MAb) (MR1, 0.2 mg i.p. daily beginning on the day of infection; Taconic) or control hamster immunoglobulin G (IgG; Rockland, Gilbertsville, PA). To stimulate CD40 signaling, mice received protein G-purified anti-CD40 MAb (1C10, 25 μg i.p. daily beginning 1 day prior to infection). A rat IgG2 MAb (GL117) was used as a control. IFN- was neutralized by administration of XMG1.2 (0.5 mg i.p. on days –1 and 3 postinfection). Depletion of T cells was accomplished by injection of anti-CD4 (GK1.5) and anti-CD8 (2.43) MAb (1 mg i.p. of each MAb) 2 days before and the day of infection. Administration of these MAbs causes a 90% reduction of T cells as assessed by flow cytometry.
Peritoneal cell preparations. Peritoneal cells were collected by lavage with 2 ml of ice-cold PBS. Smears of 5 x 104 peritoneal cells per mouse were obtained using a cytocentrifuge (Cytospin; Shandon, Pittsburgh, PA). Slides were fixed and stained with Diff-Quick (Dade Diagnostics, Aguada, Puerto Rico). Five hundred cells were analyzed per sample to determine the percentages of T. gondii-infected cells, the number of tachyzoites per 100 peritoneal cells, and the phenotypic composition of cell preparations. The total number of tachyzoites per peritoneal cavity was calculated using the number of parasites per 100 peritoneal cells and the number of peritoneal cells obtained per mouse.
Macrophages and in vitro infection with T. gondii. Peritoneal macrophages obtained as described previously (1) were cultured on eight-chamber tissue culture glass slides (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) at 5 x 105 cells/ml in complete medium consisting of Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Nonadherent cells were removed after 6 h. Tachyzoites of the RH strain of T. gondii were used to infect monolayers at a ratio of 1.5 to 3 parasites per macrophage. Parasite replication was assessed by light microscopy (3). Bone marrow-derived macrophages were obtained as described previously (1).
Induction of CD154 expression. Purified CD4+ T cells were obtained from IFN-–/– mice 7 days after i.p. infection with 1 x 103 tachyzoites of the ts4 strain of T. gondii. Uninfected IFN-–/– mice were used as controls. Spleen cells from these animals were incubated with anti-CD4-coated magnetic beads (Miltenyi Biotec, Auburn, CA), and rosetting cells were removed with a magnet. Populations of CD4+ T cells were >95% CD4+. CD4+ T cells (5 x 105/ml) and autologous bone marrow-derived macrophages (1.25 x 105/ml) were cultured in 96-well plates containing complete medium. For stimulation with T. gondii, CD4+ T cells were incubated with macrophages challenged with T. gondii tachyzoites at a multiplicity of infection of 4 tachyzoites per macrophage as previously described (36). CD154 induction was assessed at 18 h (predetermined optimal time point for detection of CD154).
Flow cytometry. T cells were incubated with Fc block reagent (BD Biosciences, San Jose, CA), followed by staining with anti-CD3 peridinin chlorophyll protein, anti-CD4 fluorescein isothiocyanate (FITC), and anti-CD154 phycoerythrin and appropriate isotype control MAbs (BD Biosciences). Cells were fixed with 1% paraformaldehyde and were analyzed using a FACSCalibur (BD Biosciences). Expression of CD154 was analyzed on 2 x 104 CD4+ T cells.
For detection of intracellular TNF-, peritoneal cells were incubated in complete medium with or without T. gondii-DsRed (1 tachyzoite per peritoneal cell) or with IFN- (100 U/ml; Peprotech, Rocky Hill, NJ) plus lipopolysaccharide (LPS) (100 ng/ml; Sigma Chemical, St. Louis, MO). Cells were incubated in the presence of brefeldin A (10 μg/ml; Sigma Chemical) for 6 h, followed by staining with either F4/80-FITC (Serotec, Raleigh, NC) or appropriate isotype control MAb. Cells were fixed and permeabilized by using IntraPrep permeabilization reagent (Coulter-Immunotech, Hialeah, FL) according to the manufacturer's instructions. Thereafter, cells were stained with anti-TNF--allophycocyanin or appropriate isotype control MAbs (BD Biosciences). After fixation with 1% paraformaldehyde, cells were analyzed using a FACSCalibur. Forward and side scatter properties as well as F4/80 expression were used to identify macrophages. Expression of DsRed was used to identify infected cells. Expression of intracellular TNF- was analyzed in infected and uninfected macrophages.
Immunofluorescence. To determine whether cell-associated T. gondii-DsRed was intracellular, peritoneal cells incubated with the parasite were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were incubated with blocking buffer without prior permeabilization, followed by incubation with anti-SAG1 MAb (DG52; gift from John Boothroyd). Cells were stained with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Eugene, OR) and examined using a Zeiss Axioplan equipped for epifluorescence microscopy. Intracellular tachyzoites display only red fluorescence, while extracellular ones exhibit both red and green fluorescence.
Cytokine assays. Blood was incubated at room temperature for 1 h, followed by incubation at 4°C for 1 h to induce clot retraction. Samples were spun at 140 x g for 10 min at 4°C. Serum was collected and centrifuged at 1,500 x g for 15 min at 4°C. Concentrations of IFN- in sera were determined by enzyme-linked immunosorbent assay (Endogen, Rockford, IL).
Measurement of nitrite production. Culture supernatants of macrophages were collected 24 h after incubation with stimulatory anti-CD40 or control MAb or IFN- plus LPS and 24 h after addition of T. gondii. The amount of nitrite released was calculated colorimetrically using the Griess reaction (Sigma Chemical). Data are expressed as μM nitrite.
Statistical analysis. Statistical significance was assessed by Student's t test or analysis of variance using InStat, version 3.0 (GraphPad, San Diego, CA).
RESULTS
Blockade of endogenous CD154 increases parasite load in IFN--deficient mice infected with T. gondii. We determined if endogenous CD154 plays a role in restricting the growth of T. gondii in IFN-–/– mice. These mice were infected with 1 x 103 tachyzoites of the avirulent temperature-sensitive mutant ts4 strain of T. gondii, followed by administration of control or blocking anti-CD154 MAb. As shown in Fig. 1, blockade of CD154 caused an increase in the percentage of infected peritoneal cells, the number of tachyzoites per 100 peritoneal cells, and the total number of tachyzoites per peritoneal cavity in IFN-–/– mice ([2.6 ± 0.04]-, [2.86 ± 0.17]-, and [4.3 ± 0.6]-fold increases, respectively; P < 0.02). Thus, endogenous CD40-CD154 interaction restrains in vivo growth of T. gondii independently of IFN-.
The results presented above suggested the presence of CD154 during in vivo T. gondii infection. While CD154 can be expressed by a variety of cells, activated CD4+ T cells are considered a major source of this molecule. Thus, we determined whether T. gondii infection induces expression of CD154 on CD4+ cells. Purified CD4+ T cells obtained from either uninfected or T. gondii-infected IFN-–/– mice were incubated with uninfected or T. gondii-infected autologous bone marrow-derived macrophages. As shown in Fig. 2, CD4+ T cells from T. gondii-infected IFN-–/– mice upregulated CD154 when exposed to parasite-infected macrophages (P < 0.001). Thus, T. gondii infection induces a subpopulation of CD4+ T cells that acquires expression of CD154 in response to the parasite.
In vivo CD40 stimulation decreases parasite load in T. gondii-infected IFN-–/– mice and induces STAT1-independent macrophage anti-T. gondii activity. To confirm the role of CD40 during T. gondii infection, we examined the effects of in vivo administration of a stimulatory anti-CD40 MAb. IFN-–/– mice were infected i.p. with tachyzoites of the ts4 strain of T. gondii and were treated with either stimulatory anti-CD40 or control MAb. As shown in Fig. 3, CD40 stimulation caused a marked decrease in the percentage of infected peritoneal cells, the number of tachyzoites per 100 peritoneal cells, and the total number of tachyzoites per peritoneal cavity (52.3% ± 3.1%, 72.4% ± 5.1%, and 77.6% ± 4.0% inhibition, respectively; P < 0.02; n = 3). The effects of anti-CD40 MAb were similar in mice infected with tachyzoites of the ts4 strain of T. gondii or tissue cysts of the ME49 strain of the parasite (data not shown).
Next, we began to explore the mechanism(s) by which in vivo CD40 ligation reduces parasite load in T. gondii-infected mice. CD40 ligation on antigen-presenting cells can modulate T-cell function (19). Thus, we examined whether T cells mediate reduction in parasite load caused by in vivo administration of anti-CD40 MAb. Depletion of T cells did not alter reduction in the percentage of infected peritoneal cells, the number of tachyzoites per 100 peritoneal cells, and the total number of tachyzoites per peritoneal cavity caused by CD40 stimulation (60.1% ± 4.6%, 69.5% ± 5.0%, and 81.0% ± 7.1% reduction, respectively; P < 0.03; n = 3) (Fig. 3). Thus, a stimulatory anti-CD40 MAb reduced the load of T. gondii in the absence of IFN-. In addition, events that follow CD40 engagement do not need T cells to restrict T. gondii growth.
Macrophages are a major component of the immune response against T. gondii. Therefore, we determined whether in vivo CD40 ligation results in induction of macrophage anti-T. gondii activity independently of IFN-. IFN-–/– mice received either a stimulatory anti-CD40 or control MAb i.p. Peritoneal cells were collected 24 h post-MAb administration. No significant difference in the percentage of large mononuclear cells was noted between samples obtained from mice treated with control or anti-CD40 MAb (control, 87.2% ± 3.7%; anti-CD40, 81.8% ± 1.2%; P = 0.17; n = 3). Macrophages were challenged with tachyzoites of the RH strain of T. gondii and were examined by light microscopy. While macrophages from mice that received control MAb did not exhibit anti-T. gondii activity, macrophages obtained from IFN-–/– mice treated with stimulatory anti-CD40 MAb showed a marked decrease in the infection rate from 1 h to 18 h postchallenge (58.84% ± 2.53% reduction; P = 0.0002; n = 3), indicative of killing of the parasite. In addition, macrophages from mice treated with anti-CD40 MAb had a significantly lower parasite load than control macrophages at 18 h postchallenge (65.6% ± 3.0% reduction; P < 0.0001) (Fig. 4). CD40 stimulation induced anti-T. gondii activity even in monolayers with high percentages of infected macrophages. Monolayers with a 68% infection rate at 1 h postchallenge exhibited a 52.7% ± 2.6% reduction in infection rate at 18 h. Administration of higher amounts of anti-CD40 MAb did not further increase anti-T. gondii activity of macrophages (data not shown). Thus, in vivo CD40 stimulation induces macrophage toxoplasmacidal activity independently of IFN-.
Signal transducer and activator of transcription 1 (STAT1) is a signaling molecule central for induction of macrophage antimicrobial activity and resistance against T. gondii (7, 15, 27). STAT1 is required for induction of NOS2 and p47 GTPases (LRG-47 and IGTP) in macrophages (7, 15, 18), effector molecules that control macrophage antimicrobial activity and mediate resistance against T. gondii (8, 32, 42). Thus, we determined whether in vivo CD40 ligation requires STAT1 to restrict T. gondii growth. STAT1–/– mice infected with tachyzoites of the ts4 strain of T. gondii were treated with either control or stimulatory anti-CD40 MAb. As shown in Fig. 5, CD40 stimulation induced a marked reduction (69.7 ± 4.6%; n = 3; P = 0.01) in parasite load in STAT1–/– mice. In agreement with the restriction of T. gondii growth induced by CD40 in STAT1–/– mice, production of reactive nitrogen intermediates, LRG-47, and IGTP did not mediate anti-T. gondii activity. Peritoneal macrophages obtained from IFN-–/– mice treated in vivo with control or anti-CD40 MAb did not produce nitrite when cultured in vitro with or without T. gondii (<5 μM; n = 3). In contrast, macrophages from both groups of mice produced nitrite in response to in vitro culture with IFN-/LPS (24.9 ± 1.3 mM; n = 3). Finally, CD40 stimulation of bone marrow macrophages from LRG-47–/–, IGTP–/–, IRG-47–/–, and wild-type mice resulted in a similar reduction in parasite load (wild type, 32.9% ± 1.5%; LRG-47–/–, 35.7% ± 4.0%; IGTP–/–, 38.8% ± 4.6%; IRG-47–/–, 39.9% ± 1.6%; P = 0.2; n = 3). Taken together, STAT1, reactive nitrogen intermediates, LRG-47, IGTP, and IRG-47 are not required for CD40 stimulation to restrict T. gondii growth in macrophages.
CD40 ligation modulates TNF- production. In vitro studies revealed that autocrine TNF- is required for induction of toxoplasmacidal activity following CD40 stimulation (1). We examined the effect of in vivo CD40 ligation on TNF- production using flow cytometry and assessment of intracellular levels of this cytokine. Preliminary studies revealed that cells sorted from a macrophage gate as determined by forward and side scatter properties were >93% large mononuclear cells with <4% granulocytes. Next, we used a combination of forward and side scatter properties plus expression of the surface marker F4/80 to study TNF- production. Peritoneal cells from mice treated in vivo with control or anti-CD40 MAb were infected with transgenic T. gondii that express DsRed in the cytoplasm. Staining with antibody against the parasite antigen SAG-1 plus Alexa Fluor 488-conjugated secondary antibody of nonpermeabilized peritoneal cells followed by examination using an epifluorescence microscope revealed that >82% of cell-associated T. gondii was intracellular. Thus, expression of DsRed by flow cytometry was used to identify infected cells. Infection with T. gondii did not affect the expression of intracellular TNF- in F4/80+ macrophages from mice treated with control MAb (Fig. 6) (P = 0.6; n = 3). In contrast, in vivo CD40 ligation resulted in increased production of TNF- in T. gondii-infected F4/80+ macrophages (Fig. 6) (P = 0.01; n = 3). Thus, in vivo CD40 stimulation enhances TNF- production in T. gondii-infected peritoneal macrophages.
TNFR2 is required for CD40-induced reduction of parasite load in IFN--depleted mice. CD40 synergizes with TNFR2 but not TNFR1 to induce anti-T. gondii activity in macrophages (2). We determined if in vivo reduction in parasite load triggered by CD40 stimulation requires the presence of TNFR2. B6 or TNFR2–/– mice were treated with a neutralizing anti-IFN- MAb, followed by infection with tachyzoites of the ts4 strain of T. gondii. No detectable levels of IFN- were noted in sera from T. gondii-infected mice that received anti-IFN- MAb. In addition, while mice treated with a control MAb did not exhibit detectable tachyzoites in peritoneal cavities, parasites were readily noted in IFN--depleted mice (Fig. 7). IFN--depleted B6 and TNFR2–/– mice infected with T. gondii were treated with either stimulatory anti-CD40 or control MAbs. While the stimulatory anti-CD40 MAb caused a marked decrease (81.7 ± 6.4%; P = 0.01) in parasite load in IFN--depleted B6 mice, this effect was not noted in IFN--depleted TNFR2–/– mice (P = 0.8). Thus, TNFR2 is required for CD40 stimulation to reduce the load of T. gondii in IFN--depleted hosts.
DISCUSSION
The immune response against pathogens such as T. gondii is multifaceted. While IFN- is central for control of the parasite, many lines of evidence strongly suggest that there are additional mechanisms of control of T. gondii. Despite the fact that IFN-–/– mice are extremely susceptible to T. gondii, even when infected with a nonvirulent strain of the parasite, we report that the CD40-CD154 pathway restrains parasite proliferation in these mice. In vivo CD40 stimulation decreases parasite load in these mice, while blockade of endogenous CD40-CD154 interaction markedly increases parasite load. These findings are important because they may identify an alternate arm of cell-mediated immunity that acts in concert with IFN- for optimal control of T. gondii, explaining why IFN- appears to be insufficient for control of infection in CD154–/– and TNFR1/2–/– mice (30, 44). Given that the interaction between CD40-CD154 is crucial for resistance against numerous other pathogens, these findings raise the possibility that the CD40-CD154 pathway controls IFN--independent host protection against other pathogens besides T. gondii.
While the CD40-CD154 pathway regulated parasite load in T. gondii-infected IFN-–/– mice, we did not observe a consistent effect of this pathway on survival. These findings are compatible with the fact that mice are extremely susceptible to T. gondii in the absence of IFN- and with the notion that the CD40-CD154 pathway may act in concert with IFN- to enhance resistance against the parasite (3, 30). In apparent contrast to mice, humans with congenital defects in IFN- signaling are not susceptible to toxoplasmosis (24). The findings presented here are of importance because they may explain why these individuals control T. gondii infection.
CD40 signaling controls numerous aspects of cell-mediated immunity that include stimulation of type-1 cytokine response, T-cell priming, and modulation of macrophage antimicrobial activity (19). These effects are likely instrumental in mediating resistance to intracellular pathogens. Our results indicate that CD40 stimulation can restrain in vivo growth of T. gondii independently of IFN- and T cells. Induction of toxoplasmacidal activity in macrophages accompanies in vivo reduction in parasite load induced by CD40 stimulation. This finding suggests that modulation of macrophage function contributes to host protection against intracellular pathogens triggered by CD40.
STAT1 is a key regulator of NOS2 and p47 GTPases (7, 15, 18), effector molecules that mediate control of intracellular pathogens (8, 42). In addition to NOS2 (1), we report that CD40 ligation does not require the presence of STAT1 and the p47 GTPases LRG-47, IGTP, and IRG-47 to restrict the growth of T. gondii. These findings support the contention that CD40 modulates macrophage antimicrobial activity not only independently of IFN- but also independently of a signaling molecule and effector molecules classically controlled by this cytokine.
While TNF- is crucial for resistance against T. gondii during the chronic phase of infection, its role is dispensable during the acute phase of infection (10, 44). In contrast, we find that TNF- signaling is necessary for CD40 stimulation to restrict parasite expansion in acutely infected IFN-–/– mice. These findings are compatible with the evidence that TNF- signaling takes center stage in host protection when IFN--dependent mechanisms of resistance are deficient (40, 41, 45). In addition, this report provides an in vivo correlate to our in vitro studies where we report that CD40 synergizes with TNFR2 to induce macrophage anti-T. gondii activity (2).
T. gondii is a poor inducer of TNF- production by macrophages (3, 5). Moreover, T. gondii can actively block TNF- production by macrophages through manipulation of proinflammatory signaling cascades (12). However, the observation that T. gondii infection induces TNF- production in vivo and the role of macrophages as a source of this cytokine suggest that there are immune events that take place during T. gondii infection that upregulate macrophage production of TNF-. We report that in vivo CD40 ligation increases TNF- production by T. gondii-infected macrophages. The increase in production of this cytokine by macrophages is likely sufficient to trigger toxoplasmacidal activity in macrophages, since low concentrations of TNF- fully synergize with CD40 to induce this effector response (2). Taken together, these findings suggest that CD40 is an important modulator of TNF- signaling during T. gondii infection.
Activated CD4+ T cells trigger anti-T. gondii activity in macrophages through CD40-CD154 interaction (1). The demonstration that T. gondii infection induces parasite-reactive T cells that acquire CD154 expression provides a source of CD154 for CD40-dependent restriction of T. gondii growth. However, while activated CD4+ T cells are considered a major source of CD154, other cells (eosinophils, basophils, mast cells, and platelets) can also express functional CD154 (13, 14, 21). The fact that blockade of endogenous CD154 during the early phase of infection with T. gondii remarkably enhances parasite load raises the possibility that other cells in addition to CD4+ T cells may also activate CD40-dependent mechanisms of control of parasite replication.
The CD40-CD154 pathway is an important regulator of type 1 cytokine response explaining in part why defects in this pathway lead to susceptibility to opportunistic infections. Using a murine model of T. gondii infection, we report that the CD40-CD154 pathway restrains the growth of an intracellular pathogen in IFN--deficient hosts. These findings provide evidence of an unappreciated role of CD40-CD154 signaling in the in vivo immune response to intracellular pathogens.
ACKNOWLEDGMENTS
We thank Gregory Taylor for providing us with macrophages from p47 GTPase-deficient mice and John Boothroyd for making anti-SAG1 MAb available for these studies.
This work was supported by National Institutes of Health grant AI48406 and the American Heart Association, Ohio Valley Affiliate (C.S.S.).
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