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Infection and Immunity, August 2004, p. 4552-4560, Vol. 72, No. 8
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.8.4552-4560.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A STAT4-Dependent Th1 Response Is Required for Resistance to the Helminth Parasite Taenia crassiceps

Miriam Rodríguez-Sosa,1,2 Rafael Saavedra,3 Eda P. Tenorio,3 Lucia E. Rosas,4 Abhay R. Satoskar,4* and Luis I. Terrazas1,2*

Department of Immunology, Instituto Nacional de Cardiología "Ignacio Chávez," México D.F. 14080,1 Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonoma de Mexico, México D.F. 04510,3 Unidad de Biomedicina, FES-Iztacala, Universidad Nacional Autonoma de Mexico, Estado de México 54090, México,2 Department of Microbiology, The Ohio State University, Columbus, Ohio 432104

Received 3 September 2003/ Returned for modification 15 December 2003/ Accepted 28 April 2004


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ABSTRACT
 
To determine the role of STAT4-dependent Th1 responses in the regulation of immunity to the helminth parasite Taenia crassiceps, we monitored infections with this parasite in resistant mice lacking the STAT4 gene. While T. crassiceps-infected STAT4+/+ mice rapidly resolved the infection, STAT4–/– mice were highly susceptible to infection and displayed large parasite loads. Moreover, the inability of STAT4–/– mice to control the infection was associated with the induction of an antigen-specific Th2-type response characterized by significantly higher levels of Th2-associated immunoglobulin G1 (IgG1) and total IgE as well as interleukin-4 (IL-4), IL-10, and IL-13 than those in STAT4+/+ mice, who produced significantly more gamma interferon. Furthermore, early after infection, macrophages from STAT4–/– mice produced lower levels of the pro-inflammatory cytokines IL-12, tumor necrosis factor alpha, IL-1ß, and nitric oxide (NO) than those from STAT4+/+ mice, suggesting a pivotal role for macrophages in mediating protection against cysticercosis. These findings demonstrate a critical role for the STAT4 signaling pathway in the development of a Th1-type immune response that is essential for mediating protection against the larval stage of T. crassiceps infection.


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INTRODUCTION
 
Neurocysticercosis is a life-threatening helminth infection caused by the larvae of the cestode Taenia solium, which infects humans and pigs. Although cysticercosis is considered an important public health problem in South America and Asia, recent studies have shown that this disease can also affect populations in developed countries (36, 38). Cysticercosis caused by Taenia crassiceps usually affects rodents, with canines as final hosts (10), although there are reports that immunocompromised humans can be infected with this parasite (21). Nevertheless, experimental murine cysticercosis has been a useful model for studying the immune mechanisms involved in determining the disease outcome of cysticercosis (30, 47).

It has been widely accepted that a Th2-mediated response plays a critical role in the host defense against helminth infections (11, 22), primarily those caused by gastrointestinal nematodes (48). However, several reports have shown that some stages of helminth parasites are not efficiently controlled by specific Th2-type responses. For example, one study found that a Th2 response was not required for controlling Brugia malayi infection (16). Furthermore, others reported that an early interleukin-12 (IL-12)-dependent Th1 response is necessary to mediate vaccine-induced protection against schistosomiasis (25).

A series of studies found that although T. crassiceps-infected BALB/c mice develop an initial but brief Th1-like response, it is replaced by a strong Th2-biased response that is in turn associated with an increase in the parasite load (46, 51). Members of our laboratory showed previously that the administration of anti-gamma interferon (IFN-{gamma}) neutralizing antibodies to T. crassiceps-infected mice during the early phase of infection renders them more susceptible to cysticercosis (43). Similarly, it was also found that IL-12 p35–/– BALB/c mice are more susceptible to the larval stage of T. crassiceps (33). Conversely, T. crassiceps-infected STAT6–/– mice mounted a strong Th1 response in the absence of Th2 development and controlled the infection (31). Taken together, these observations indicate that while a Th1-type response and IFN-{gamma} are essential for the development of immunity against experimental cysticercosis, Th2-type responses may have a limited role in the control of this parasitic infection.

Several studies have shown that IFN-{gamma} production, which is required for immunity against T. crassiceps, can be induced via both STAT4-dependent and STAT4-independent signaling pathways (6, 27). Therefore, to determine the relative roles of STAT4-dependent and STAT4-independent signaling pathways in the development of protective immunity against cysticercosis, we compared the course of T. crassiceps infection in genetically resistant C57BL/6 x 129Sv/Ev mice lacking the STAT4 gene with that in their age- and sex-matched wild-type (STAT4+/+) counterparts. In addition, we analyzed the antigen-specific antibody profiles in sera, the cellular immune responses, and cytokine profiles in both spleen cells and peritoneal macrophages as well as the kinetics of cellular recruitment at the site of infection.

Our data demonstrate that the STAT4-dependent IL-12 signaling pathway is essential for the development of immunity against cysticercosis.


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MATERIALS AND METHODS
 
Animals. STAT4–/– mice were generated by gene disruption as previously described (44). The STAT4-KO mice used for our experiments were obtained from homozygous inbreeding in the F2 generation (129Sv x C57BL/6). Additionally, we also performed an experiment with STAT4–/– and STAT4+/+ littermates derived by intercrossing of the third-generation STAT4+/– mice backcrossed to the C57BL/6 strain, and we used C57BL/6 mice (Harlan, Mexico) as an additional control (Fig. 1, inset). The mice in all experiments were 8 to 10 weeks old and were bred and maintained in the specific-pathogen-free facility at the Instituto Nacional de Cardiología "Ignacio Chávez" according to the Institutional and National Guidelines for Animal Research.



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  		FIG. 1. STAT4–/– mice are highly susceptible while STAT4+/+ mice are highly resistant to T. crassiceps infection. The graph shows the course of T. crassiceps infection in STAT4–/– and STAT4+/+ mice after i.p. infection with 20 cysticerci. Data are expressed as mean parasite loads ± standard errors (SE) of six mice per group. *, P < 0.01 for STAT4–/– versus STAT4+/+ data at the same time point. The data shown are representative of two independent experiments. Inset, C57BL/6 and STAT4+/+ littermates displayed similar resistance patterns, while partially backcrossed STAT4–/– mice were highly susceptible to T. crassiceps infection. *, P < 0.05 for STAT4–/– versus STAT4+/+ and C57BL/6 mice (five to six mice per group).

Parasites and infection protocols. Metacestodes of T. crassiceps were harvested from the peritoneal cavity of female BALB/c mice after 2 to 4 months of infection. The cysticerci were washed four times in sterile phosphate-buffered saline (PBS) (0.15 M, pH 7.2). Experimental infection was achieved by intraperitoneal (i.p.) injection with 20 small (ca. 2 mm in diameter) nonbudding cysticerci of T. crassiceps suspended in 0.3 ml of PBS per mouse, and mice were sacrificed at weeks 2, 4, 8, and 16 postinfection. The parasite load was evaluated by counting all parasites found in the peritoneal cavity after extensive washes with PBS.

T. crassiceps soluble antigen (TcAg) was obtained from freshly and sterilely isolated cysticerci from BALB/c female mice after 2 to 4 months of infection. The parasites were extensively washed with PBS and homogenized with a Tissue Tearor (Dremel, Racine, Wis.) for cycles of 2 to 3 min on ice. Homogenized cysticerci were centrifuged at 10,000 rpm for 1 h at 4°C, the supernatant was collected, and protein levels were determined by the Bradford method. TcAg for cell cultures was sterilized by filtration.

Cell preparations, culture conditions, and cytokine assays. Proliferation assays were performed on spleen cells obtained from T. crassiceps-infected mice at different time points after infection. Briefly, single-cell suspensions were prepared in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U of penicillin-streptomycin, 2 mM glutamine, 25 mM HEPES buffer, and 1% nonessential amino acids (complete medium) (all from GIBCO BRL, Grand Island, N.Y.). The erythrocytes were lysed, and viable cells were adjusted to 3 x 106 cells/ml. The cell suspension (100 µl/well) was placed into 96-well flat-bottomed culture plates (Costar, Cambridge, Mass.), stimulated with TcAg (50 µg/ml) in a total volume of 200 µl, and incubated at 37°C for 96 h. Eighteen hours prior to culture termination, 0.5 µCi of [3H]thymidine (185 GBq/mmol) (Amersham, Buckinghamshire, England) per well was added. The cells were harvested and thymidine uptake was measured with a Betaplate counter (Wallac, Turku, Finland). Values are expressed as mean counts per minute for triplicate wells and are the results after subtracting the counts per minute for cultures in the absence of antigen. The supernatants from these cultures were analyzed for IFN-{gamma}, IL-4, IL-10 (PharMingen, San Diego, Calif.), and IL-13 (R&D Systems, Minneapolis, Minn.) production by enzyme-linked immunosorbent assays (ELISAs).

Isolation of CD4+ T cells and cultures with CD4-depleted splenocytes. Spleen cells were depleted of CD4+ T cells (>95% by fluorescence-activated cell sorting [FACS] analysis) by the use of CD4 magnetic cell sorter beads (MACS; Miltenyi Biotec, Bergisch, Germany) according to the manufacturer's instructions. CD4– splenocytes (<5% CD4+) (3 x 106 cells/ml; 100 µl/well) were plated in 96-well flat-bottomed plates (Costar) as described above. The cultures were maintained at 37°C in 5% CO2 for 4 days, and then [3H]thymidine (Amersham) (0.5 µCi/well) was added and the cells were incubated for a further 18 h. The cells were harvested on a 96-well harvester (Tomtec, Toku, Finland) and counted with a Betaplate counter. Similar cultures were performed with enriched CD4+ cells (2 x 106 cells/ml; 100 µl/well) from the same mice, using irradiated splenocytes from healthy STAT4+/+ and STAT4–/– mice as antigen-presenting cells (APC) (106 cells/ml; 100 µl/well), and the cultures were processed as described above.

Isolation of peritoneal macrophages and analysis of response to LPS-plus-IFN-{gamma} stimulation. Peritoneal exudate cells (PECs) were obtained from the peritoneal cavity of mice infected with T. crassiceps at 2, 4, 8, and 16 weeks postinfection. The cells were washed twice with Hanks balanced salt solution, and erythrocytes were lysed by resuspending the cells in Boyle's solution (0.17 M Tris and 0.16 M ammonium chloride). After two more washes, viable cells were counted by trypan blue exclusion. PECs were adjusted to 5 x 106/ml in complete RPMI and were cultured in six-well plates (Costar). After 2 h at 37°C in 5% CO2, nonadherent cells were removed by washing with warm supplemented RPMI medium. Cold Ca2+- and Mg2+-free PBS was added, the cells were incubated for 5 min, and adherent cells were gently detached with a sterile rubber policeman. The plates were rinsed twice with Ca2+- and Mg2+-free PBS for the collection of residual cells. These cells were centrifuged and readjusted to 106/ml. Viability was determined by trypan blue exclusion and was usually >95%. These cells constituted >90% macrophages according to FACS analysis with the F4/80 monoclonal antibody. One milliliter of cell suspension was then plated, and cell activation was performed in 24-well plates (Costar) with lipopolysaccharide (LPS) (1 µg/ml, from Escherichia coli 111:B4; Sigma, St Louis, Mo.) plus 2 ng of recombinant murine IFN-{gamma} (BD Pharmingen, San Diego, Calif.)/ml followed by incubation for 24 h at 37°C in 5% CO2. The supernatants were harvested, centrifuged, and examined by ELISAs for IL-1-ß, IL-12, IL-18, and tumor necrosis factor alpha (TNF-{alpha}) production (antibodies and cytokines were obtained from BD Pharmingen) and for nitric oxide (Griess reaction) production. Total PECs were also analyzed by a cytospin preparation stained with Wright-Giemsa stain (Sigma), and 300 cells were counted per slide.

Determination of proliferation of CD4 and CD8 T cells by CFSE staining. Spleen cells (107 cells) were stained with 0.5 µM CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) as previously described (34). One million cells were stimulated with TcAg (50 µg/ml) in a total volume of 2 ml in 24-well plates and were incubated at 37°C in 5% CO2 for 4 days. The cells were harvested, washed with Dulbecco's PBS-1% fetal calf serum-0.1% NaN3, and stained for 30 min (4°C) with a phycoerythrin (PE)-labeled anti-CD4 monoclonal antibody (1 µg/106 cells) from clone RM4-5 (BD Biosciences) or with a PE-labeled anti-CD8 monoclonal antibody (0.25 µg/106 cells) from clone 53-6.7 (BioLegend, San Diego, Calif.). The cells were washed twice in the same buffer, resuspended in Dulbecco's PBS, and analyzed by flow cytometry. Events were captured as previously described (23).

Flow cytometric analysis. The expression of membrane markers on peritoneal adherent cells was analyzed by flow cytometry. Adherent PECs were blocked with an anti-mouse Fc{gamma}R antibody (CD16/CD32) and stained with fluorescein isothiocyanate-conjugated monoclonal antibodies against F4/80 (Serotec, Oxford, United Kingdom) or PE-conjugated antibodies against CD23 or CCR5 (BD Pharmingen). Stained cells were analyzed on a FACSCalibur cytometer (Becton Dickinson, Mountain View, Calif.).

Antibody ELISA. Blood was collected from the tails of T. crassiceps-infected STAT4+/+ and STAT4–/– mice at different times after infection. Specific end-point titers of immunoglobulin G1 (IgG1) and IgG2a were determined by ELISA as previously described (42). Total IgE production (serum dilution, 1:10) was detected by Opt-ELISA (BD Pharmingen).

Statistical analysis. Comparisons between groups were made with Student's t test. P values of <0.05 were considered significant. The statistical significance of the titers in sera was determined by a nonparametric Mann-Whitney U-Wilcoxon rank test.


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RESULTS AND DISCUSSION
 
It is widely accepted that the Th2-like response plays a critical role in mediating protective immunity against most helminthic infections (11). For example, either IL-4 or IL-13 is necessary to expulse the gastrointestinal nematode Trichuris muris (5) and the STAT6-mediated signaling pathway has also been shown to promote protective immunity against Trichinella spiralis (50) and Nippostrongylus brasiliensis (49). Nevertheless, some studies have reported that the Th2-type response may not be effective against certain stages of helminth parasites (3, 39). In fact, some investigators, including us, have shown that an IL-12-induced Th1-like response is necessary for the successful control of helminths such as Schistosoma mansoni (4) and T. crassiceps (33). For the present study, we used STAT4–/– mice to investigate the potential roles of the STAT4-dependent and STAT4-independent signaling pathways in the development of a protective Th1 response during T. crassiceps infection. As early as 2 weeks after i.p. inoculation with 20 nonbudding cysticerci, striking differences in parasite numbers were observed between STAT4–/– mice (20 ± 4 parasites) and STAT4+/+ (1 ± 1 parasites) mice (Fig. 1). Furthermore, as infection progressed, the parasite burdens increased dramatically in STAT4–/– mice compared to in STAT4+/+ mice, which successfully controlled parasite growth at 2 weeks postinfection and contained only a few parasites in their peritoneal cavities (Fig. 1). Similar results were also observed in experiments that were performed with 10 and 25 cysticerci infecting the mice (data not shown). Together, these findings indicate that a STAT4-mediated IL-12 signaling pathway plays a critical role in the development of protective immunity against cysticercosis caused by T. crassiceps. Furthermore, in an independent experiment we also found that STAT4–/– mice derived by the intercrossing of STAT4+/– mice were significantly more susceptible to T. crassiceps than were their STAT4+/+ littermates, suggesting that the phenotype observed for STAT4–/– mice is most likely due to the selective lack of STAT4 rather than other genes.

Several studies have demonstrated that a STAT4-mediated IL-12 signaling pathway mediates protective immunity against intracellular protozoan parasites by favoring Th1 development and simultaneously inhibiting the development of a detrimental Th2 response (7, 40, 41). In contrast, only one study on the role of STAT4 during helminth infections has been performed which demonstrated that STAT4–/– mice develop smaller pulmonary granulomas after schistosome egg injection (14). We had previously shown that susceptible mice treated with IFN-{gamma} plus IL-2 during the early course of T. crassiceps infection restricted parasite growth, suggesting that the Th1 response mediates protective immunity against this parasite (43). In the present study, by week 4 postinfection and thereafter, T. crassiceps-infected STAT4–/– mice displayed significantly higher titers of Th2-associated TcAg-specific IgG1 (Fig. 2a) and total IgE (Fig. 2c) but significantly less Th1-associated TcAg-specific IgG2a (at 8 and 16 weeks postinfection) than similarly infected STAT4+/+ mice (Fig. 2b). Furthermore, at weeks 2, 4, 8, and 16 postinfection, TcAg-stimulated spleen cells from STAT4+/+ mice produced markedly elevated and sustained levels of IFN-{gamma} compared to those from STAT4–/– mice, who produced significantly more IL-10 at week 4 postinfection and thereafter (Fig. 3a and b). At all of these time points, TcAg-stimulated spleen cells from T. crassiceps-infected STAT4–/– mice secreted persistently higher levels of IL-4 and IL-13 which peaked at week 8 postinfection (Fig. 3c and d).



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  		FIG. 2. Kinetics of antibody production during T. crassiceps infection of STAT4–/– (closed circles) and STAT4+/+ (open circles) mice. (a) Anti-T. crassiceps IgG1. (b) Anti-T. crassiceps IgG2a. (c) Total IgE. The graphs show mean titers ± SE (n = 8 animals) and are representative of two independent experiments. *, P < 0.05 for STAT4–/– versus STAT4+/+ data at the same time point.



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  		FIG. 3. Kinetics of in vitro cytokine production by TcAg-stimulated spleen cells from STAT4–/– and STAT4+/+ mice. (a) IFN-{gamma}; (b) IL-10; (c) IL-4; (d) IL-13. The graphs show cytokine production by splenocytes after 72 h of in vitro stimulation with TcAg (50 µg/ml).

Interestingly, the spleen cells from T. crassiceps-infected STAT4+/+ and STAT4–/– mice displayed differences in the magnitudes of proliferative responses after antigen-specific in vitro recall. While TcAg-stimulated splenocytes from STAT4+/+ and STAT4–/– mice displayed comparable proliferative responses during early phases of infection, as infection progressed those from STAT4+/+ mice displayed significantly higher responses than those from STAT4–/– mice (Fig. 4). To ensure that this discrepancy in proliferation between STAT4+/+ and STAT4–/– mice was not due to differences in the numbers of CD4+ cells, we determined the proportions of CD4+ and CD8+ T cells in the spleens by flow cytometry. As previously reported for T. crassiceps-infected BALB/c mice (42), no significant differences were found in the percentages of CD4+ and CD8+ T cells between STAT4+/+ and STAT4–/– mice (data not shown). Furthermore, TcAg-specific proliferative responses observed in spleen cells from T. crassiceps-infected mice were abolished by the depletion of CD4+ T cells, suggesting that the main subpopulation of cells proliferating in response to TcAg was the CD4+ T cells (Fig. 4a). Moreover, when we analyzed the TcAg-specific proliferative response by CFSE and CD4/CD8-PE labeling, we confirmed that the predominant T-cell population proliferating in response to antigenic stimulation was CD4+ T cells (Fig. 4b). Nevertheless, both CD4+ and CD8+ T cells displayed a higher proliferative response in wild-type mice than in STAT4–/– mice. These data support observations in previous studies of cysticercosis and other helminthic diseases showing that the chronic stage of infection is characterized by down-regulated immune cell responses to parasite antigens (37, 42, 46) and that this phenomenon is not due to a reduction in the number of CD4 T cells.




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  		FIG. 4. Cell type analysis of proliferative responses to TcAg-specific stimulation. (a) Splenocytes from STAT4–/– and wild-type mice taken 2, 4, 8, and 16 weeks after T. crassiceps infection were stimulated with 50 µg of TcAg/ml. In some experiments, CD4 cells were magnetically removed and CD4– splenocytes were stimulated in the same way as total splenocytes. *, P < 0.05 for STAT4–/– versus STAT4+/+ data at the same time points. (b) TcAg-specific proliferation of CD4 and CD8 cells. Spleen cells from infected wild-type and STAT4–/– mice (at 2 and 16 weeks postinfection) were labeled with CFSE and cultured with 50 µg of TcAg/ml for 4 days prior to staining with a PE-conjugated anti-CD4 or anti-CD8 monoclonal antibody and analysis on a flow cytometer. Five thousand nonproliferating, CFSE+ (CD4+ or CD8+) cells were collected, and the percentages of proliferating and nonproliferating cells were calculated and are indicated in each dot plot. The results shown are representative of those obtained for three mice per group.

Despite the lower magnitudes of their proliferative responses, TcAg-stimulated spleen cells from STAT4–/– mice produced significantly higher levels of Th2-type cytokines than did STAT4+/+ mice, indicating that the low proliferation detected in STAT4–/– infected mice does not necessarily reflect less cytokine production, but rather a differential pattern of secretion. These findings are similar to that reported for the murine model of filariasis (2). Another interesting observation in our study was that T. crassiceps-infected STAT4+/+ mice efficiently controlled parasite burdens despite producing significantly lower levels of total IgE. These observations suggest a limited role for IgE in mediating protective immunity against T. crassiceps, although they do not exclude a role for antigen-specific IgE in the control of cysticercosis. Although Th2-associated IgE has been largely shown to play a role in mediating immunity to helminths (8, 9), one study has found that the lack of IgE does not alter immunity to S. mansoni (39). Nevertheless, taken together, these findings demonstrate that the Th1-type response induced via the STAT4-dependent signaling pathway is essential for the control of cysticercosis, whereas a Th2-type response is detrimental and enhances susceptibility to the disease. Consistent with our observations, other studies using a radiation-attenuated vaccine with IL-12 as an adjuvant have also shown that a Th1-like response can mediate protective immunity against certain parasitic helminths, such as Schistosoma (26).

Importantly, we found that CD4+ cells were the main source of the cytokines analyzed (Table 1), with the exception of IL-10 in STAT4–/– mice, in which CD4-depleted spleen cells maintained the capacity to produce IL-10, suggesting that cells other than CD4+ T cells may be a possible source of this cytokine which remain to be identified. A crucial role for IL-10 in the regulation of immunity has been reported for other helminthic diseases, such as schistosomiasis (35) and filariasis (20). Moreover, we have previously shown that a blockade of IL-10 in susceptible BALB/c mice improved their ability to control cysticercosis (43). Thus, IL-10 may play a major role in the pathogenesis of cysticercosis due to its immunomodulatory activities, such as the down-regulation of costimulatory molecules on APC or the suppression of IL-12, IFN-{gamma}, and other pro-inflammatory cytokines (24).


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  		TABLE 1. Antigen-specific cytokine production by CD4-depleted and CD4-enriched splenocytes from STAT4+/+ and STAT4–/– mice infected with T. crassicepsa

Several studies have demonstrated that macrophages play a critical role in immunity against many intracellular pathogens by their ability to secrete Th1-inducing cytokines such as IL-12 and IL-18 and to produce nitric oxide (NO), which is microbicidal even to larvae of Schistosoma or Brugia (45). We have hypothesized that macrophages may be involved in mediating protective immunity against T. crassiceps metacestodes in STAT6–/– mice by secreting Th1-inducing cytokines such as IL-12 and/or by releasing NO. Therefore, we analyzed IL-1ß, IL-12, IL-18, TNF-{alpha}, and NO production by adherent peritoneal macrophages from T. crassiceps-infected STAT4+/+ and STAT4–/– mice. LPS-plus-IFN-{gamma}-activated macrophages from T. crassiceps-infected STAT4+/+ mice obtained during the early phase of infection (2 weeks) produced higher levels of IL-1ß, TNF-{alpha}, and IL-12 than those from similarly infected STAT4–/– mice (Fig. 5a to c). As infection progressed, macrophages from chronically infected STAT4+/+ mice produced lower levels of IL-1ß, IL-18, and IL-12 (Fig. 5a, c, and d), but significantly higher levels of TNF-{alpha} (Fig. 5b). This pattern of macrophage response was different from that observed for STAT4–/– mice, who displayed a low production level of IL-12, IL-1ß, and TNF-{alpha} early in infection but had a sustained production of IL-18 during the later phase of infection (Fig. 5a to d). Macrophages from T. crassiceps-infected STAT4–/– mice also produced significantly lower levels of NO than those from STAT4+/+ mice throughout the course of infection (Fig. 5e). Taken together, these observations suggest that the STAT4 signaling pathway mediates resistance to T. crassiceps, at least in part, by favoring macrophage IL-1ß and TNF-{alpha} production, which could be involved directly or indirectly in parasite elimination. It is also likely that the impaired NO production observed for STAT4–/– macrophages is due to their inability to produce TNF-{alpha}, which is known to activate macrophages to produce NO. The roles of these factors in regulating resistance to cysticercosis will be the focus of future investigations in our laboratory.



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  		FIG. 5. Peritoneal macrophages from STAT4–/– (closed circles) and STAT4+/+ (open circles) T. crassiceps-infected mice display different responses after in vitro stimulation with LPS (1 µg/ml) plus IFN-{gamma} (2 ng/ml) for 24 h. IL-1ß (a), TNF-{alpha} (b), IL-12 (c), IL-18 (d), and NO (e) production by macrophages at different weeks after T. crassiceps infection was determined by ELISA or the Griess reaction. Data are the means ± standard deviations for six animals at each time point. *, P < 0.05 for STAT4–/– versus STAT4+/+ data at the same time points.

Classically, eosinophils have been considered to be among the most efficient effector cells in several helminth parasitic diseases (8, 9). Therefore, we also evaluated the proportions of cell populations in inflammatory infiltrates at the site of infection (peritoneal cavity) in STAT4–/– and STAT4+/+ mice at different time points after T. crassiceps infection. At 2 weeks postinfection, STAT4–/– mice recruited >12% eosinophils into their peritoneal cavities, and this high percentage was maintained throughout the course of infection (Fig. 6a). In contrast, the peritoneal cavities of STAT4+/+ mice contained only a few eosinophils (<2%; P < 0.05) (Fig. 6a). Nonetheless, STAT4+/+ mice had an early increased infiltration of macrophages and lymphocytes compared to STAT4–/– mice (Fig. 6b to c). Additional differences were observed in the recruitment of neutrophils, which were detected in higher percentages in the peritoneal cavities of STAT4–/– mice during the early course of infection (Fig. 6d). No significant difference was observed in the numbers of basophils/mast cells between the groups. These results suggest that eosinophils, neutrophils, and basophils/mast cells do not play a significant role in mediating resistance against T. crassiceps infection and oppose the dogma that eosinophils are the key cells that play a critical role in eliminating helminths (8, 9). However, further studies are warranted to evaluate the definitive role of eosinophils in immunity against cysticercosis by using IL-5–/– mice, who fail to develop blood or tissue eosinophilia (15). Nevertheless, our data favor a possible active participation of macrophages in eliminating T. crassiceps, possibly by producing NO. In fact, we have also found that an enhanced resistance of STAT6–/– and CD40–/– mice to T. crassiceps infection is associated with a significant increase in NO production by macrophages (31, 33). Moreover, in support of this view, it is known that the sustained production of NO enhances the cytostatic or cytotoxic activity of macrophages against viruses, bacteria, fungi, protozoa, tumor cells (19), and even helminths such as B. malayi and S. mansoni (1, 45).



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  		FIG. 6. T. crassiceps infection recruits significantly larger numbers of eosinophils to the site of infection in STAT4–/– mice. (a) Percentages of eosinophils; (b) percentages of macrophages; (c) percentages of lymphocytes; (d) percentages of neutrophils; (e) percentages of basophils/mast cells. The graphs show the recruitment of cells to the peritoneal cavity after T. crassiceps infection. Mice were infected i.p. with 20 cysticerci and were sacrificed at the indicated times. The composition of cells was analyzed after a cytospin and Wright-Giemsa staining, with at least 300 cells counted per slide. Analyses were performed in individual mice (six mice per group). The data are representative of two independent experiments. *, P < 0.05 for STAT4–/– versus STAT4+/+ data at the same time points.

Recently, several groups have reported a different class of macrophages in helminth infections (17, 28), known as "alternatively activated" macrophages (12). In order to determine whether the enhanced susceptibility of STAT4–/– mice to T. crassiceps was associated with an increase in the number of this type of macrophages, we analyzed the expression of alternatively activated macrophage markers CD23 and CCR5 on peritoneal macrophages from T. crassiceps-infected STAT4+/+ and STAT4–/– mice (12, 32). Interestingly, while macrophages from resistant STAT4+/+ mice did not express CD23 and presented a low level of expression of CCR5, those from STAT4–/– mice displayed significantly higher expression levels of both CD23 and CCR5 (Fig. 7). It is noteworthy that CCR5 expression in CD4 and CD8 cells has been shown to be dependent on the IL-12- and STAT4-mediated signaling pathway (13). However, our findings in the present study suggest that CCR5 expression in macrophages is STAT4 independent. It is also known that chemokines can downmodulate the expression of their own receptors after binding (18). Hence, it is likely that the high level of expression of CCR5 observed on macrophages from T. crassiceps-infected STAT4–/– mice is due to a low level of production of the chemokine CCL5, which binds CCR5. In fact, a recent study in an allergy model showed that STAT4–/– mice have a reduced production of several chemokines, included CCL5 (29).



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  		FIG. 7. T. crassiceps infection up-regulates the expression of CD23 and CCR5 in peritoneal macrophages from STAT4–/– mice. Peritoneal macrophages from STAT4+/+ and STAT4–/– infected mice were obtained at different time points after infection and stained with anti-F4/80 in combination with anti-CD23-PE, anti-CCR5-PE, or an isotype control antibody. The histograms shown were gated on the F4/80+ adherent peritoneal cells (dotted line, isotype; thick line, STAT4–/– mice; dashed line, STAT4+/+ mice). The data are representative of two independent experiments, with three to four mice per group.

In conclusion, genetically resistant C57BL/6 x 129Sv/Ev mice that are partially backcrossed onto C57BL/6 mice and that lack a STAT4-dependent IL-12 signaling pathway fail to mount an efficient Th1 response, develop a Th2 response, and become highly susceptible to the helminth parasite T. crassiceps. Moreover, the susceptibility of STAT4–/– mice to T. crassiceps appears to be associated with the inability of their macrophages to produce adequate amounts of pro-inflammatory cytokines and nitric oxide. These results demonstrate that the STAT4-dependent signaling pathway is required for the development of resistance to murine cysticercosis and that a STAT4-independent pathway alone is not sufficient to confer protection against this infection.


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ACKNOWLEDGMENTS
 
We thank Carlos A. Tena and Veronica Graullera for their excellent assistance with the care of STAT4–/– and STAT4+/+ mice.

This work was supported by CONACYT grant 41584-M and by PAPCA-FES-Iztacala, UNAM. A.R.S. was supported by a grant from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address for Luis I. Terrazas: Department of Immunology, Instituto Nacional de Cardiología "Ignacio Chávez," J. Badiano # 1, México, D.F. 14080, México. Phone: (5255) 5573-2911. Fax: (5255) 5573-0994. E-mail: terlui{at}cardiologia.org.mx. Mailing address for Abhay R. Satoskar: Department of Microbiology, The Ohio State University, 484 West 12th Ave., Columbus, OH 43221. Phone: (614) 292-3243. Fax: (614) 292-8120. E-mail: satoskar0.2{at}osu.edu. Back

Editor: J. F. Urban, Jr.


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Infection and Immunity, August 2004, p. 4552-4560, Vol. 72, No. 8
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.8.4552-4560.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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