Macrophage Migration Inhibitory Factor Contributes to Host Defense against Acute Trypanosoma cruzi Infection
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感染与免疫杂志 2006年第6期
Unidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autonoma de Mexico (UNAM) Tlalnepantla
Instituto de Investigaciones Biomedicas, UNAM
Instituto Nacional de Cardiología Ignacio Chávez, Mexico D.F., Mexico
Department of Microbiology, The Ohio State University, Columbus, Ohio
ABSTRACT
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is involved in the host defense against several pathogens. Here we used MIF–/– mice to determine the role of endogenous MIF in the regulation of the host immune response against Trypanosoma cruzi infection. MIF–/– mice displayed high levels of blood and tissue parasitemia, developed severe heart and skeletal muscle immunopathology, and succumbed to T. cruzi infection faster than MIF+/+ mice. The enhanced susceptibility of MIF–/– mice to T. cruzi was associated with reduced levels of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-12 (IL-12), IL-18, gamma interferon (IFN-), and IL-1, in their sera and reduced production of IL-12, IFN-, and IL-4 by spleen cells during the early phase of infection. At all time points, antigen-stimulated splenocytes from MIF+/+ and MIF–/– mice produced comparable levels of IL-10. MIF–/– mice also produced significantly less Th1-associated antigen-specific immunoglobulin G2a (IgG2a) throughout the infection, but both groups produced comparable levels of Th2-associated IgG1. Lastly, inflamed hearts from T. cruzi-infected MIF–/– mice expressed increased transcripts for IFN-, but fewer for IL-12 p35, IL-12 p40, IL-23, and inducible nitric oxide synthase, compared to MIF+/+ mice. Taken together, our findings show that MIF plays a role in controlling acute T. cruzi infection.
INTRODUCTION
Chagas' disease is caused by the flagellate protozoon Trypanosoma cruzi, which is transmitted by the feces of blood-sucking insect vectors (Triatoma). The parasite plays a fundamental role in inducing immunopathology and tissue damage in organs such as the heart, esophagus, and colon by sequentially inducing inflammatory responses, cellular lesions, and fibrosis (60). Host resistance in experimental acute T. cruzi infection is dependent on both innate and acquired immune responses mediated by macrophages, natural killer (NK) cells, CD4+ T cells, CD8+ T cells, and B cells (17, 31, 33). Proinflammatory cytokines, such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-), and gamma interferon (IFN-), also play a critical role in protective immunity against T. cruzi (40, 44, 48, 59), since genetically engineered mice lacking any of these cytokines fail to control parasitemia and rapidly succumb to infection. IL-12 mediates immunity to T. cruzi infection by enhancing IFN- production from NK cells, which is required for the induction of NO-dependent macrophage microbicidal activity as well as the subsequent induction of a protective Th1 response (21, 24, 30, 40). On the other hand, the anti-inflammatory cytokines IL-4 and IL-10 mediate susceptibility to T. cruzi, but IL-10 is also required for preventing immune hyperactivity and organ immunopathology (22, 23).
Migration inhibitory factor (MIF) is a pleiotropic cytokine produced by activated T cells (5, 13), macrophages (8), and the pituitary gland (4). While MIF is involved in the pathogenesis of several inflammatory diseases, such as chronic colitis (14, 47), rheumatoid arthritis (41), and atherosclerosis (29), it is also a key effector molecule in modulating protective immunity against bacteria (10, 28, 46, 54), protozoan parasites (26, 37, 55), and helminths (51, 56). The protective role of MIF during microbial infection has been attributed to its ability to initiate an innate immune response by inducing TNF- production (7, 11, 16), up-regulating Toll-like receptor 4 (TLR4) expression (52), and enhancing macrophage microbicidal activity (26). While some previous studies had suggested that MIF plays a critical role in T-cell activation and cytokine production, we and others have found that MIF-deficient T cells proliferate as efficiently as wild-type (WT) T cells following in vitro antigenic or mitogenic stimulation and produce significant amounts of Th1/Th2-associated cytokines comparable to those in WT mice, indicating that a lack of MIF has no clear effect on T-cell activity (51, 55).
The present study was undertaken to examine the in vivo role of MIF in determining the outcome of experimental Chagas' disease caused by T. cruzi. Our results demonstrate that MIF plays an important role in the host defense against acute T. cruzi infection by favoring production of the proinflammatory cytokines IL-12, IL-18, TNF-, IL-1, and IFN- during the early phase of infection. Moreover, they also show that increased susceptibility of MIF–/– mice to T. cruzi is not due to poor T-cell activation or a compensatory increase in the production of anti-inflammatory cytokines.
MATERIALS AND METHODS
Mice. Eight- to 10-week-old male BALB/c mice were purchased from Harlan (Mexico City, Mexico) and maintained in a pathogen-free environment at our animal facility in accordance with institutional and national guidelines. MIF–/– mice were developed as described previously and backcrossed for >10 generations to a BALB/c genetic background (7).
Parasites and infections. The Mexican Trypanosoma cruzi isolate Ninoa (nonpathogenic) was obtained from a human patient in the acute phase of disease and used in part of this work (43). This isolate belongs to the T. cruzi I lineage and was maintained by sequential culture in liver infusion tryptose medium (Difco, Detroit, MI), Triatoma pallidipennis infection, and murine passages as described previously (18). Mice were inoculated by subcutaneous injection (1 x 103 metacyclic trypomastigotes) into the hind footpad. The Mexican T. cruzi isolate Queretaro (pathogenic) was obtained from a human patient in the acute phase of disease and was maintained by sequential murine passages. This isolate belongs to the T. cruzi I lineage (6). Blood trypomastigotes were obtained from previously infected mice, counted, and adjusted to 5 x 104/ml in sterile phosphate-buffered saline (PBS), and 100 μl was injected intraperitoneally (i.p.) into male MIF–/– and MIF+/+ mice. Parasitemia was determined by using hemocytometer counts of parasites in blood diluted 1:10 in PBS with 3.8% sodium citrate.
Trypanosoma cruzi lysate antigen. Briefly, epimastigotes of T. cruzi that had been maintained by sequential culture in liver infusion tryptose medium were isolated, washed three times in PBS, and centrifuged at 1 x 104 rpm for 15 min. Protease inhibitors were added (0.1 to 2 μg/ml aprotinin, 0.5 to 2 mM EDTA, 1 to 5 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 50 μg/ml TLCK [N-p-tosyl-L-lysine chloromethyl ketone] [all from Sigma, St. Louis, MO]), and parasites were then sonicated six times for 10 s each time at 50 W, using a Sonic Dimembrator 300 instrument (Fisher). Parasite destruction was determined using a microscope. Parasite lysates were then centrifuged at 1 x 104 rpm for 30 min to separate out the soluble fraction, which was stored at –70°C until use. The protein concentration was determined by the Lowry method (35).
Cell preparations, culture conditions, and cytokine assays. Spleen cells were obtained and cultured as previously described (51). Briefly, single-cell suspensions were prepared in complete RPMI 1640 supplemented with 10% fetal bovine serum, 100 units of penicillin-streptomycin, 2 mM glutamine, 25 mM HEPES buffer, and 1% nonessential amino acids (all from GIBCO BRL, Grand Island, NY). Erythrocytes were lysed, and viable cells were adjusted to 3 x 106 cells/ml. Cell suspensions (100 μl/well) were plated in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and stimulated with T. cruzi lysate antigen (25 μg/ml) for 72 h at 37°C. Supernatants from these cell cultures and sera obtained from infected MIF+/+ and MIF–/– mice were analyzed to determine the production of IL-18, IL-12, IFN-, IL-4, IL-10 (all from Pharmingen, San Diego, CA), IL-1, and TNF- (Peprotech, Mexico) by enzyme-linked immunosorbent assays (ELISAs) performed according to the manufacturers' instructions.
Antibody ELISAs. Peripheral blood was collected at 5-day intervals from tail snips of T. cruzi-infected MIF–/– and MIF+/+ BALB/c mice. Blood was centrifuged at 200 x g, and sera were collected and tested for T. cruzi lysate-specific immunoglobulin G1 (IgG1) and IgG2a contents. Briefly, 96-well polystyrene ELISA plates (Corning, Corning, NY) were coated with 100 μl/well (5 μg/ml) of T. cruzi lysate antigen diluted in PBS, pH 9.0, and incubated overnight at 4°C. Plates were washed using PBS with 0.05% Tween 20 (PBS-Tween 20; Sigma, St. Louis, MO) and were blocked with PBS supplemented with 1% bovine serum albumin for 1 h at 37°C. Serially diluted serum samples (1/100 starting dilution in PBS-Tween 20) were added to the plates in duplicate and incubated for 2 h at 37°C. Bound antibodies were detected by incubation with either horseradish peroxidase-conjugated rat anti-mouse IgG1 or horseradish peroxidase-conjugated rat anti-mouse IgG2a (diluted 1/5,000 in 25% bovine serum and 75% PBS; Zymed, San Francisco, CA). After incubation for 1 h, the plates were washed, developed with ABTS solution (Zymed), and read on a microplate reader at 405 nm (Multiskan Ascent; Thermo Labsystems). Results are expressed as mean end-point titers ± standard errors (SE).
Histopathology. Hearts and skeletal muscle from legs were fixed overnight in formaldehyde and embedded in paraffin blocks, after which 5-μm-thick transverse sections were mounted on slides and subsequently stained with hematoxylin and eosin. Using an Olympus BX51 microscope (Olympus American, Melville, NY) equipped with a digital video camera, 25 individual fields were evaluated per sample for each tissue, covering at least 80% of the total area of the section. The severity of inflammation was evaluated as the number of positive inflammatory cells per field, and the numbers of amastigote nests were counted in the same sections by two pathologists.
RT-PCR analysis. Total RNA was isolated from the hearts of MIF+/+ and MIF–/– T. cruzi-infected mice at 22 days postinfection by the TRIzol method (GIBCO BRL) according to the manufacturer's instructions. cDNA samples were amplified by SuperScript One-Step reverse transcription-PCR (RT-PCR) for 25 to 30 cycles, using Platinum Taq polymerase (Invitrogen) and specific primers (the sequences of the primers used are given in Table 1). After amplification, PCR products were separated by electrophoresis on 1.5% agarose gels containing SYBR green I, a nucleic acid gel stain used at 1,000x (Amresco), and were visualized with the FLA-5000 chemiluminescence detection system (Fujifilm). The data were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using the Multi Gauge Image program. Statistical analysis was performed using Student's t test. All reactions were repeated two times to ensure reproducibility.
Relative gene expression by real-time quantitative PCR. IL-23 (p19), IL-12 p35, IL-12 p40, and inducible nitric oxide synthase (iNOS) mRNA expression in the hearts of MIF+/+ and MIF–/– mice at 22 days post-T. cruzi infection was determined by RT, followed by analysis of relative gene expression by real-time quantitative PCR and the 2 – CT method (34). Briefly, total RNA of heart cells was extracted using TRIzol reagent (GIBCO BRL) according to the manufacturer's instructions. RT-PCR was performed using an Access RT-PCR system kit according to the manufacturer's protocol (Promega). The p19, p35, p40, and iNOS primers were labeled with SYBR green I (1x) (Amresco) and processed as described elsewhere (49). Real-time PCR was performed on a Rotor Gene RG 3000 instrument (Corbett Research, Australia). As a control, GAPDH housekeeping gene expression was evaluated using the same PCR protocol as that for p19, p35, p40, and iNOS. The specificities of PCRs for p19, p35, p40, and iNOS were verified by the absence of a signal in no-template controls for the heart samples. The cycle threshold (CT) was used to reflect the relative p19, p35, p40, and iNOS mRNA expression levels and to calculate the change in gene expression between infected MIF–/– and MIF+/+ mice. Data evaluation and processing were performed according to the methods of Ponchel et al. (49). The sequences of the primers used were as follows: for IL-23 (p19), 5'-TGC TGG ATT GCA GAG CAG TAA-3' and 5'-CTG GAG GAG TTG GCT GAG TC-3' (3); for IL-12 (p35), 5'-GCA AGA GAC ACA GTC CTG GG-3' and 5'-TGC ATC AGC TCA TCG ATG GC-3' (61); for IL-12 (p40), 5'-GGA GAC CCT GCC CAT TGA ACT-3' and 5'-CAA CGT TGC ATC CTA GGA TCG-3' (3); and for iNOS, 5'-CTG GAG GAG CTC CTG CCT CATG-3' and 5'-GCA GCA TCC CCT CTG ATG GTG-3' (63).
Statistical analysis. Comparisons between the MIF+/+ and MIF–/– groups were made by using Student's unpaired t test. P values of <0.05 were considered significant. For survival assays, a log-rank test was used with the GraphPad computer program (GraphPad, San Diego, CA). The statistical significance of serum titers was determined by using the nonparametric Mann-Whitney U-Wilcoxon rank sum test.
RESULTS
MIF–/– mice develop high parasitemia levels and show increased mortality when infected with nonpathogenic and pathogenic strains of T. cruzi. MIF is required for initial IL-12 and TNF- production by macrophages in response to lipopolysaccharide-mediated septic shock and to Salmonella infection (9, 28, 36). To further investigate the role of MIF in immunity to T. cruzi, MIF–/– and MIF+/+ mice were infected with two different strains of T. cruzi. One strain, Ninoa, which is nonpathogenic (according to its inability to produce tissue lesions and mortality), was injected into the footpad at a low dose of 1 x 103 metacyclic trypomastigotes obtained from the urine of the intermediate host Triatoma, and parasitemia was monitored. T. cruzi-infected MIF–/– mice developed significantly higher blood parasitemia levels than similarly infected MIF+/+ mice, which contained nearly threefold fewer parasites in their blood by days 22 to 30 postinfection (p.i.) (Fig. 1A) (P < 0.05).
Independent experiments were performed using the pathogenic strain (Queretaro) of T. cruzi, with MIF–/– and MIF+/+ mice being infected by i.p. inoculation of 5 x 103 blood trypomastigotes. Both groups developed blood parasitemia on day 10 p.i., but on day 16 p.i., MIF–/– mice displayed significantly greater levels of parasitemia, which peaked at day 27 p.i. and was positively detected until day 44 p.i. (Fig. 1B). In contrast, the maximum peak of parasitemia in MIF+/+ mice was observed on day 22 p.i., and these mice controlled the infection by day 30. Furthermore, MIF–/– mice also started to succumb to infection as early as day 18 p.i., and just 30% of them survived to day 45. In contrast, 70% of MIF+/+ mice survived throughout the infection (Fig. 1C).
IL-12 and IFN- responses are impaired in MIF–/– mice during early T. cruzi infection. IL-12 and IFN- have largely been associated with protection during acute T. cruzi infection (1, 24, 30, 33, 57). Antigen-presenting cells are the major source of innate IL-12 production in response to T. cruzi and its products (12). On the other hand, besides NK cells, T cells are a major source of IFN- (58). To determine whether the increased susceptibility and mortality of MIF–/– mice correlated with altered production of these cytokines, we compared IL-12 and IFN- levels in the sera of T. cruzi-infected MIF+/+ and MIF–/– mice as early as 3 days p.i. until 30 days p.i. After infection with the pathogenic strain of T. cruzi, MIF+/+ mice displayed rapid production of IL-12 and IFN- (Fig. 2A and B), which was sustained, or in some cases increased, for at least 3 weeks after infection. In contrast, MIF–/– mice displayed a significant impairment in the production of these cytokines early in the infection (Fig. 2A and B). As infection progressed, however, increased levels of IL-12 and IFN- were detected in the sera of MIF–/– mice. Consistent with these in vivo data, spleen cells from MIF+/+ infected animals produced more IL-12 and IFN- in response to T. cruzi lysate antigens than did MIF–/– mice in early infections. Similarly, by day 22 p.i., both in vitro groups produced comparable levels of these cytokines (Fig. 2C and D).
Proinflammatory cytokine production is altered in MIF–/– mice after T. cruzi infection. Other potent proinflammatory cytokines, such as TNF-, IL-1, and IL-18, have been shown to play a role in determining the outcome of T. cruzi infection (25, 40, 44). The main sources of the early production of these cytokines are macrophages and dendritic cells. While it is known that macrophage TNF- secretion is up-regulated by MIF (26), the role of MIF in regulating IL-1 and IL-18 remains elusive. To determine whether the production of proinflammatory cytokines is influenced by MIF during T. cruzi infection, we measured the levels of TNF-, IL-1, and IL-18 in the sera of MIF–/– and MIF+/+ mice during the course of infection. TNF- was detected in sera from both groups early during the course of infection, but the levels were significantly higher in MIF+/+ mice than in MIF–/– mice (Fig. 3A). However, as the infection progressed, both MIF+/+ and MIF–/– mice displayed comparable levels of TNF- in their sera. Similarly, TNF- levels in spleen and lymph node cell culture supernatants were also elevated in WT mice during the early phase of infection (data not shown). Interestingly, the kinetics of IL-1 and IL-18 production also displayed similar patterns, and serum levels of both of these cytokines were significantly higher in MIF+/+ mice than in MIF–/– mice throughout the course of the infection (Fig. 3B and C). These data suggest a pivotal role for MIF in regulating IL-1 and IL-18 production.
Augmented Th2-type reactions are not responsible for impaired proinflammatory responses in MIF–/– mice. Since Th2-type cytokines, such as IL-4 and IL-10, are involved in both susceptibility to T. cruzi infection and down-regulation of inflammatory cytokines (23, 30), we determined whether altered levels of these cytokines were responsible for the enhanced susceptibility of MIF–/– mice to acute T. cruzi infection. No significant differences were noted in the serum levels of IL-4 and IL-10 between T. cruzi-infected WT and MIF–/– mice at all time points examined throughout the course of the infection (Fig. 4A and B). The production of T. cruzi lysate-specific IL-4 by spleen cells from MIF–/– mice was lower early in the infection, whereas IL-10 production was similar between groups (Fig. 4C and D). Thus, it appears that elevated levels of Th2-type cytokines are unlikely to be responsible for the down-regulation of proinflammatory cytokine production observed in MIF–/– mice.
T. cruzi-infected MIF–/– mice develop severe heart and skeletal muscle pathologies. On day 22 p.i., the hearts and skeletal muscles of MIF+/+ mice showed low tissue parasitism with moderate inflammatory mononuclear cell infiltration but without major histopathological signs of lesions (Fig. 5A and C). In contrast, histopathology sections of infected MIF–/– mice showed an intense inflammatory reaction in the heart tissue (Fig. 5B and E), with a marked increase in amastigote nests (Fig. 5F) which was accompanied by severe heart injury due to large necrotic lesions. Damage was also more evident in the skeletal muscles of MIF–/– infected mice, which had large lesions and a larger number of amastigote nests than those of MIF+/+ T. cruzi-infected mice (Fig. 5C and D). Nevertheless, the heart was the more severely affected tissue, with larger numbers of amastigote nests and greater inflammatory infiltrates than the skeletal muscles of MIF–/– mice (Fig. 5E and F). These results suggest that MIF–/– mice succumb to the classical pathophysiology of the infection, which is typical of a severe acute phase of experimental Chagas' disease.
Analysis of IFN-, CCL5, IL-12 p35, IL12 p40, p19 (IL-23), and iNOS mRNA expression in infected hearts. Since MIF–/– mice displayed severe cardiac pathology, we examined the cytokine and chemokine expression in this organ. On day 22 after T. cruzi infection, total mRNA was isolated from hearts, and RT-PCR analyses were performed. As shown in Fig. 6A and B, infected hearts from MIF–/– mice displayed a significant increase in transcripts of IFN-, but similar levels for CCL5, compared to hearts from MIF+/+ mice. Furthermore, transcripts of IFN- were detectable in MIF–/– mice by using amounts of cDNA as low as 7 ng (Fig. 6A), while using this amount of cDNA yielded barely detectable IFN- transcripts from MIF+/+ mice. Interestingly, -actin transcripts were more intensely detected in MIF–/– T. cruzi-infected mice. Using 1 μg of mRNA, similar -actin intensities were detected for both groups (data not shown); however, when dilutions of heart mRNA were performed, it was evident that MIF–/– infected mice expressed -actin more intensely than MIF+/+ infected mice (Fig. 6A and B). This apparent lack of good quality control is explained by the known effects of intense T. cruzi infection on heart muscle cells, where the disruption of the actin cytoskeleton generates an increase in -actin production (39). This observation is consistent with the higher parasite burdens and severe pathology in the infected hearts of MIF–/– mice. In contrast, when another housekeeping mRNA was tested (GAPDH), no differences in the transcripts were detected between MIF–/– and MIF+/+ infected mice (Fig. 6A). Additionally, using real-time RT-PCR, we measured IL-12, IL-23, and iNOS mRNA levels that are likely involved in mediating the host defense against T. cruzi. As shown in Fig. 6C, T. cruzi-infected MIF+/+ mice showed significant increases in the levels of IL-12 p35, IL-12 p40, IL-23 p19, and iNOS mRNAs in their hearts compared to similarly infected MIF–/– mice.
IFN--dependent, antigen-specific IgG2a antibody titers are decreased in MIF–/– mice. IFN- and anti-T. cruzi specific antibodies have been shown to act synergistically in protecting mice against this parasite (31, 40). Given that IFN- is known to modulate IgG production, particularly that of IgG2a, and that IFN- levels were similar in late infections between MIF–/– and MIF+/+ mice, we decided to evaluate the kinetics of parasite-specific IgG1 and IgG2a antibodies during T. cruzi infection. Throughout the course of infection, T. cruzi-infected MIF–/– mice displayed lower titers of T. cruzi-specific IgG2a than did MIF+/+ mice, and the differences were statistically significant on days 21 and 28 p.i. (Fig. 7A), despite the fact that the levels of IFN- in both the sera and spleen cell supernatants were comparable between groups at these time points. In contrast, T. cruzi-specific IgG1 production was not severely affected by MIF deficiency (Fig. 7B).
DISCUSSION
Our study shows that MIF–/– mice are highly susceptible to experimental Chagas' disease caused by T. cruzi. Infection of MIF–/– mice with 5 x 103 blood trypomastigotes of the pathogenic T. cruzi strain (Queretaro) resulted in prolonged and high parasitemia levels, severe cardiac immunopathology, and increased mortality. MIF–/– mice also developed relatively high blood parasitemia levels and cardiac inflammation when infected with 1 x 103 metacylic promastigotes of the nonpathogenic strain of T. cruzi, but they eventually resolved the infection as efficiently as MIF+/+ mice. These findings indicate that MIF plays a role in the host defense against acute T. cruzi infection.
The proinflammatory cytokines IL-12, IFN-, and TNF- play an important role in mediating resistance to T. cruzi infection (24, 44, 59), and MIF promotes IL-12 and TNF- production in macrophages (11, 26). Furthermore, MIF also mediates host immunity against intracellular pathogens by activating macrophages and enhancing their microbicidal activity by increasing NO production (26). In the present study, we found that T. cruzi-infected MIF–/– mice produced significantly less IL-12, IL-18, IL-1, and TNF- during the early phase of infection. Furthermore, we also found that hearts from T. cruzi-infected MIF–/– mice contained significantly less iNOS mRNA than hearts from MIF+/+ mice. Taken together, these findings suggest that endogenous MIF is required for optimal production of proinflammatory cytokines and NO during the early phase of T. cruzi infection. Because MIF–/– mice produce significantly less NK-cell-activating IL-12 and IL-18, the lack of IFN- production early in infection in these mice can be explained by an inadequate activation of NK cells, which are by far the most important source of IFN- at early stages of T. cruzi infection (40). It is important that the numbers of NK cells and macrophages were not diminished in MIF–/– infected mice, as measured by flow cytometry (data not shown). However, the requirement of MIF for efficient production of IL-1, IL-18, and IL-12 in innate immunity against T. cruzi infection appears to be a new finding.
The major pathway reported for MIF-induced proinflammatory cytokine production has been attributed to its ability to regulate lipopolysaccharide responses through the modulation of TLR4 expression on macrophages (53). Interestingly, TLR4-mediated responses to T. cruzi have been reported as one of the main pathways for inducing early cytokine production associated with innate immunity (48). Thus, low expression of TLR4 may also be involved in the low inflammatory response observed in MIF–/– T. cruzi-infected mice.
It is known that at early stages of acute T. cruzi infection, cell infiltrates are necessary to control parasite replication; however, excessive infiltration can be dangerous for the host. In the present study, the hearts from T. cruzi-infected MIF–/– mice expressed low levels of proinflammatory cytokines, such as IL-12 and IL-23, and the enzyme iNOS, which is responsible for the production of nitric oxide, an effector molecule involved in killing of T. cruzi (21). IL-23 has recently been associated with the promotion of autoimmune diseases (32), but its role in infectious diseases is still unknown. To our knowledge, this is the first time that a lack of IL-23 expression has been associated with MIF deficiency. Nonetheless, we also found that MIF–/– mice develop intense cardiac inflammation despite the lack of strong proinflammatory cytokine production. This controversial observation can be explained by the fact that the infected hearts of MIF–/– and MIF+/+ mice displayed similar expression levels of CCL5, which facilitates the trafficking of leukocytes to the heart and favors the development of an inflammatory infiltrate. In fact, a recent study showed that mice lacking CCR5, a receptor for CCL5, develop high parasitemia levels and cardiac parasitism and show reduced survival following T. cruzi infection (19). In our study, we also found that infected hearts from MIF–/– mice contained significantly more IFN- than those from wild-type mice, which is likely to contribute to cardiac immunopathology. Nonetheless, MIF–/– mice were unable to clear the parasites from the hearts despite intense cardiac inflammation, suggesting that the cardiac damage may be due to the large numbers of amastigote nests instead of immunity-mediated damage caused by inflammation. Although the mechanism by which MIF contributes to protective host immunity against T. cruzi is not entirely clear, perhaps the activation of infiltrating cells is adversely affected in the absence of MIF signaling, resulting in poor effector function and reduced killing of parasites.
A previous study demonstrated that MIF plays a critical role in the activation of T cells (2). However, adaptive immunity appears not to be affected by MIF deficiency, since T. cruzi antigen-stimulated spleen cells from both MIF+/+ and MIF–/– mice proliferated similarly and produced comparable levels of Th1- and Th2-type cytokines 3 weeks after infection. Thus, impaired proinflammatory cytokine production cannot be attributed to excess production of IL-4 or IL-10, both of which are known to have anti-inflammatory effects and to participate in susceptibility to T. cruzi infection. These data are consistent with previous reports using MIF–/– mice for other parasitic models (51, 55). In fact, those studies demonstrated that the absence of MIF prevents the development of protective immunity against another intracellular parasite, Leishmania major, as well as the extracellular parasite Taenia crassiceps, by inhibiting macrophage microbicidal activity rather than preventing the development of Th1-like responses. Moreover, for both parasitic infections, higher levels of antigen-specific IFN- were detected in MIF–/– mice which failed to mediate host resistance. Thus, the finding that T. cruzi lysate-specific IFN- production was not impaired in MIF–/– mice late in infection was not unexpected. It is significant, however, that despite the similar levels of circulating IFN- and the increase in mRNA expression of IFN- in cardiac tissue, MIF–/– mice displayed a deficit in T. cruzi lysate-specific IgG2a production. Interestingly, a recent report using an asthma model with MIF–/– mice showed a similar finding, in which specific anti-OVA IgG2a antibodies were significantly reduced compared with those in MIF+/+ mice (42). In line with this, the absence of MIF could affect the ability of macrophages and B cells to respond to IFN-, and hence IFN- produced by MIF–/– mice may fail to induce the efficient macrophage activation and NO production required to eliminate T. cruzi. The reason for this lack of response remains unknown but can partially explain the lack of parasite clearance by MIF–/– mice.
The pathogenicity of T. cruzi strains has been associated with the expression of some virulence factors, such as trans-sialidases (50) and the molecule gp83, that parasites use to invade cells (62). Weakly invasive strains, such as Ninoa (20), either express low levels of these markers or do not express them at all. Thus, these strains are handicapped in the ability to rapidly invade target cells, and the host may have a longer opportunity to eliminate these parasites—through both innate and adaptive mechanisms, such as complement activation and specific antibodies—before they cause tissue damage. Hence, it is likely that a less intense immune response in MIF–/– mice is sufficient to control the nonpathogenic strains of T. cruzi.
In conclusion, our findings show that endogenous MIF is involved in host resistance against acute T. cruzi infection; the higher susceptibility of MIF–/– mice may involve mechanisms that impair innate cytokine production, suggesting that MIF participates in the activation of innate immunity to induce an early inflammatory cytokine response against T. cruzi infection.
ACKNOWLEDGMENTS
This work was supported by DGAPA-PAPIIT-UNAM grant IN212804 to M.R.S. and grant IN226402 to B.E. and by an NIH grant to A.R.S.
We thank Ignacio Martínez, Benito S. Sánchez, and Rodrigo Velázquez for their technical assistance.
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Instituto de Investigaciones Biomedicas, UNAM
Instituto Nacional de Cardiología Ignacio Chávez, Mexico D.F., Mexico
Department of Microbiology, The Ohio State University, Columbus, Ohio
ABSTRACT
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is involved in the host defense against several pathogens. Here we used MIF–/– mice to determine the role of endogenous MIF in the regulation of the host immune response against Trypanosoma cruzi infection. MIF–/– mice displayed high levels of blood and tissue parasitemia, developed severe heart and skeletal muscle immunopathology, and succumbed to T. cruzi infection faster than MIF+/+ mice. The enhanced susceptibility of MIF–/– mice to T. cruzi was associated with reduced levels of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-12 (IL-12), IL-18, gamma interferon (IFN-), and IL-1, in their sera and reduced production of IL-12, IFN-, and IL-4 by spleen cells during the early phase of infection. At all time points, antigen-stimulated splenocytes from MIF+/+ and MIF–/– mice produced comparable levels of IL-10. MIF–/– mice also produced significantly less Th1-associated antigen-specific immunoglobulin G2a (IgG2a) throughout the infection, but both groups produced comparable levels of Th2-associated IgG1. Lastly, inflamed hearts from T. cruzi-infected MIF–/– mice expressed increased transcripts for IFN-, but fewer for IL-12 p35, IL-12 p40, IL-23, and inducible nitric oxide synthase, compared to MIF+/+ mice. Taken together, our findings show that MIF plays a role in controlling acute T. cruzi infection.
INTRODUCTION
Chagas' disease is caused by the flagellate protozoon Trypanosoma cruzi, which is transmitted by the feces of blood-sucking insect vectors (Triatoma). The parasite plays a fundamental role in inducing immunopathology and tissue damage in organs such as the heart, esophagus, and colon by sequentially inducing inflammatory responses, cellular lesions, and fibrosis (60). Host resistance in experimental acute T. cruzi infection is dependent on both innate and acquired immune responses mediated by macrophages, natural killer (NK) cells, CD4+ T cells, CD8+ T cells, and B cells (17, 31, 33). Proinflammatory cytokines, such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-), and gamma interferon (IFN-), also play a critical role in protective immunity against T. cruzi (40, 44, 48, 59), since genetically engineered mice lacking any of these cytokines fail to control parasitemia and rapidly succumb to infection. IL-12 mediates immunity to T. cruzi infection by enhancing IFN- production from NK cells, which is required for the induction of NO-dependent macrophage microbicidal activity as well as the subsequent induction of a protective Th1 response (21, 24, 30, 40). On the other hand, the anti-inflammatory cytokines IL-4 and IL-10 mediate susceptibility to T. cruzi, but IL-10 is also required for preventing immune hyperactivity and organ immunopathology (22, 23).
Migration inhibitory factor (MIF) is a pleiotropic cytokine produced by activated T cells (5, 13), macrophages (8), and the pituitary gland (4). While MIF is involved in the pathogenesis of several inflammatory diseases, such as chronic colitis (14, 47), rheumatoid arthritis (41), and atherosclerosis (29), it is also a key effector molecule in modulating protective immunity against bacteria (10, 28, 46, 54), protozoan parasites (26, 37, 55), and helminths (51, 56). The protective role of MIF during microbial infection has been attributed to its ability to initiate an innate immune response by inducing TNF- production (7, 11, 16), up-regulating Toll-like receptor 4 (TLR4) expression (52), and enhancing macrophage microbicidal activity (26). While some previous studies had suggested that MIF plays a critical role in T-cell activation and cytokine production, we and others have found that MIF-deficient T cells proliferate as efficiently as wild-type (WT) T cells following in vitro antigenic or mitogenic stimulation and produce significant amounts of Th1/Th2-associated cytokines comparable to those in WT mice, indicating that a lack of MIF has no clear effect on T-cell activity (51, 55).
The present study was undertaken to examine the in vivo role of MIF in determining the outcome of experimental Chagas' disease caused by T. cruzi. Our results demonstrate that MIF plays an important role in the host defense against acute T. cruzi infection by favoring production of the proinflammatory cytokines IL-12, IL-18, TNF-, IL-1, and IFN- during the early phase of infection. Moreover, they also show that increased susceptibility of MIF–/– mice to T. cruzi is not due to poor T-cell activation or a compensatory increase in the production of anti-inflammatory cytokines.
MATERIALS AND METHODS
Mice. Eight- to 10-week-old male BALB/c mice were purchased from Harlan (Mexico City, Mexico) and maintained in a pathogen-free environment at our animal facility in accordance with institutional and national guidelines. MIF–/– mice were developed as described previously and backcrossed for >10 generations to a BALB/c genetic background (7).
Parasites and infections. The Mexican Trypanosoma cruzi isolate Ninoa (nonpathogenic) was obtained from a human patient in the acute phase of disease and used in part of this work (43). This isolate belongs to the T. cruzi I lineage and was maintained by sequential culture in liver infusion tryptose medium (Difco, Detroit, MI), Triatoma pallidipennis infection, and murine passages as described previously (18). Mice were inoculated by subcutaneous injection (1 x 103 metacyclic trypomastigotes) into the hind footpad. The Mexican T. cruzi isolate Queretaro (pathogenic) was obtained from a human patient in the acute phase of disease and was maintained by sequential murine passages. This isolate belongs to the T. cruzi I lineage (6). Blood trypomastigotes were obtained from previously infected mice, counted, and adjusted to 5 x 104/ml in sterile phosphate-buffered saline (PBS), and 100 μl was injected intraperitoneally (i.p.) into male MIF–/– and MIF+/+ mice. Parasitemia was determined by using hemocytometer counts of parasites in blood diluted 1:10 in PBS with 3.8% sodium citrate.
Trypanosoma cruzi lysate antigen. Briefly, epimastigotes of T. cruzi that had been maintained by sequential culture in liver infusion tryptose medium were isolated, washed three times in PBS, and centrifuged at 1 x 104 rpm for 15 min. Protease inhibitors were added (0.1 to 2 μg/ml aprotinin, 0.5 to 2 mM EDTA, 1 to 5 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 50 μg/ml TLCK [N-p-tosyl-L-lysine chloromethyl ketone] [all from Sigma, St. Louis, MO]), and parasites were then sonicated six times for 10 s each time at 50 W, using a Sonic Dimembrator 300 instrument (Fisher). Parasite destruction was determined using a microscope. Parasite lysates were then centrifuged at 1 x 104 rpm for 30 min to separate out the soluble fraction, which was stored at –70°C until use. The protein concentration was determined by the Lowry method (35).
Cell preparations, culture conditions, and cytokine assays. Spleen cells were obtained and cultured as previously described (51). Briefly, single-cell suspensions were prepared in complete RPMI 1640 supplemented with 10% fetal bovine serum, 100 units of penicillin-streptomycin, 2 mM glutamine, 25 mM HEPES buffer, and 1% nonessential amino acids (all from GIBCO BRL, Grand Island, NY). Erythrocytes were lysed, and viable cells were adjusted to 3 x 106 cells/ml. Cell suspensions (100 μl/well) were plated in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and stimulated with T. cruzi lysate antigen (25 μg/ml) for 72 h at 37°C. Supernatants from these cell cultures and sera obtained from infected MIF+/+ and MIF–/– mice were analyzed to determine the production of IL-18, IL-12, IFN-, IL-4, IL-10 (all from Pharmingen, San Diego, CA), IL-1, and TNF- (Peprotech, Mexico) by enzyme-linked immunosorbent assays (ELISAs) performed according to the manufacturers' instructions.
Antibody ELISAs. Peripheral blood was collected at 5-day intervals from tail snips of T. cruzi-infected MIF–/– and MIF+/+ BALB/c mice. Blood was centrifuged at 200 x g, and sera were collected and tested for T. cruzi lysate-specific immunoglobulin G1 (IgG1) and IgG2a contents. Briefly, 96-well polystyrene ELISA plates (Corning, Corning, NY) were coated with 100 μl/well (5 μg/ml) of T. cruzi lysate antigen diluted in PBS, pH 9.0, and incubated overnight at 4°C. Plates were washed using PBS with 0.05% Tween 20 (PBS-Tween 20; Sigma, St. Louis, MO) and were blocked with PBS supplemented with 1% bovine serum albumin for 1 h at 37°C. Serially diluted serum samples (1/100 starting dilution in PBS-Tween 20) were added to the plates in duplicate and incubated for 2 h at 37°C. Bound antibodies were detected by incubation with either horseradish peroxidase-conjugated rat anti-mouse IgG1 or horseradish peroxidase-conjugated rat anti-mouse IgG2a (diluted 1/5,000 in 25% bovine serum and 75% PBS; Zymed, San Francisco, CA). After incubation for 1 h, the plates were washed, developed with ABTS solution (Zymed), and read on a microplate reader at 405 nm (Multiskan Ascent; Thermo Labsystems). Results are expressed as mean end-point titers ± standard errors (SE).
Histopathology. Hearts and skeletal muscle from legs were fixed overnight in formaldehyde and embedded in paraffin blocks, after which 5-μm-thick transverse sections were mounted on slides and subsequently stained with hematoxylin and eosin. Using an Olympus BX51 microscope (Olympus American, Melville, NY) equipped with a digital video camera, 25 individual fields were evaluated per sample for each tissue, covering at least 80% of the total area of the section. The severity of inflammation was evaluated as the number of positive inflammatory cells per field, and the numbers of amastigote nests were counted in the same sections by two pathologists.
RT-PCR analysis. Total RNA was isolated from the hearts of MIF+/+ and MIF–/– T. cruzi-infected mice at 22 days postinfection by the TRIzol method (GIBCO BRL) according to the manufacturer's instructions. cDNA samples were amplified by SuperScript One-Step reverse transcription-PCR (RT-PCR) for 25 to 30 cycles, using Platinum Taq polymerase (Invitrogen) and specific primers (the sequences of the primers used are given in Table 1). After amplification, PCR products were separated by electrophoresis on 1.5% agarose gels containing SYBR green I, a nucleic acid gel stain used at 1,000x (Amresco), and were visualized with the FLA-5000 chemiluminescence detection system (Fujifilm). The data were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using the Multi Gauge Image program. Statistical analysis was performed using Student's t test. All reactions were repeated two times to ensure reproducibility.
Relative gene expression by real-time quantitative PCR. IL-23 (p19), IL-12 p35, IL-12 p40, and inducible nitric oxide synthase (iNOS) mRNA expression in the hearts of MIF+/+ and MIF–/– mice at 22 days post-T. cruzi infection was determined by RT, followed by analysis of relative gene expression by real-time quantitative PCR and the 2 – CT method (34). Briefly, total RNA of heart cells was extracted using TRIzol reagent (GIBCO BRL) according to the manufacturer's instructions. RT-PCR was performed using an Access RT-PCR system kit according to the manufacturer's protocol (Promega). The p19, p35, p40, and iNOS primers were labeled with SYBR green I (1x) (Amresco) and processed as described elsewhere (49). Real-time PCR was performed on a Rotor Gene RG 3000 instrument (Corbett Research, Australia). As a control, GAPDH housekeeping gene expression was evaluated using the same PCR protocol as that for p19, p35, p40, and iNOS. The specificities of PCRs for p19, p35, p40, and iNOS were verified by the absence of a signal in no-template controls for the heart samples. The cycle threshold (CT) was used to reflect the relative p19, p35, p40, and iNOS mRNA expression levels and to calculate the change in gene expression between infected MIF–/– and MIF+/+ mice. Data evaluation and processing were performed according to the methods of Ponchel et al. (49). The sequences of the primers used were as follows: for IL-23 (p19), 5'-TGC TGG ATT GCA GAG CAG TAA-3' and 5'-CTG GAG GAG TTG GCT GAG TC-3' (3); for IL-12 (p35), 5'-GCA AGA GAC ACA GTC CTG GG-3' and 5'-TGC ATC AGC TCA TCG ATG GC-3' (61); for IL-12 (p40), 5'-GGA GAC CCT GCC CAT TGA ACT-3' and 5'-CAA CGT TGC ATC CTA GGA TCG-3' (3); and for iNOS, 5'-CTG GAG GAG CTC CTG CCT CATG-3' and 5'-GCA GCA TCC CCT CTG ATG GTG-3' (63).
Statistical analysis. Comparisons between the MIF+/+ and MIF–/– groups were made by using Student's unpaired t test. P values of <0.05 were considered significant. For survival assays, a log-rank test was used with the GraphPad computer program (GraphPad, San Diego, CA). The statistical significance of serum titers was determined by using the nonparametric Mann-Whitney U-Wilcoxon rank sum test.
RESULTS
MIF–/– mice develop high parasitemia levels and show increased mortality when infected with nonpathogenic and pathogenic strains of T. cruzi. MIF is required for initial IL-12 and TNF- production by macrophages in response to lipopolysaccharide-mediated septic shock and to Salmonella infection (9, 28, 36). To further investigate the role of MIF in immunity to T. cruzi, MIF–/– and MIF+/+ mice were infected with two different strains of T. cruzi. One strain, Ninoa, which is nonpathogenic (according to its inability to produce tissue lesions and mortality), was injected into the footpad at a low dose of 1 x 103 metacyclic trypomastigotes obtained from the urine of the intermediate host Triatoma, and parasitemia was monitored. T. cruzi-infected MIF–/– mice developed significantly higher blood parasitemia levels than similarly infected MIF+/+ mice, which contained nearly threefold fewer parasites in their blood by days 22 to 30 postinfection (p.i.) (Fig. 1A) (P < 0.05).
Independent experiments were performed using the pathogenic strain (Queretaro) of T. cruzi, with MIF–/– and MIF+/+ mice being infected by i.p. inoculation of 5 x 103 blood trypomastigotes. Both groups developed blood parasitemia on day 10 p.i., but on day 16 p.i., MIF–/– mice displayed significantly greater levels of parasitemia, which peaked at day 27 p.i. and was positively detected until day 44 p.i. (Fig. 1B). In contrast, the maximum peak of parasitemia in MIF+/+ mice was observed on day 22 p.i., and these mice controlled the infection by day 30. Furthermore, MIF–/– mice also started to succumb to infection as early as day 18 p.i., and just 30% of them survived to day 45. In contrast, 70% of MIF+/+ mice survived throughout the infection (Fig. 1C).
IL-12 and IFN- responses are impaired in MIF–/– mice during early T. cruzi infection. IL-12 and IFN- have largely been associated with protection during acute T. cruzi infection (1, 24, 30, 33, 57). Antigen-presenting cells are the major source of innate IL-12 production in response to T. cruzi and its products (12). On the other hand, besides NK cells, T cells are a major source of IFN- (58). To determine whether the increased susceptibility and mortality of MIF–/– mice correlated with altered production of these cytokines, we compared IL-12 and IFN- levels in the sera of T. cruzi-infected MIF+/+ and MIF–/– mice as early as 3 days p.i. until 30 days p.i. After infection with the pathogenic strain of T. cruzi, MIF+/+ mice displayed rapid production of IL-12 and IFN- (Fig. 2A and B), which was sustained, or in some cases increased, for at least 3 weeks after infection. In contrast, MIF–/– mice displayed a significant impairment in the production of these cytokines early in the infection (Fig. 2A and B). As infection progressed, however, increased levels of IL-12 and IFN- were detected in the sera of MIF–/– mice. Consistent with these in vivo data, spleen cells from MIF+/+ infected animals produced more IL-12 and IFN- in response to T. cruzi lysate antigens than did MIF–/– mice in early infections. Similarly, by day 22 p.i., both in vitro groups produced comparable levels of these cytokines (Fig. 2C and D).
Proinflammatory cytokine production is altered in MIF–/– mice after T. cruzi infection. Other potent proinflammatory cytokines, such as TNF-, IL-1, and IL-18, have been shown to play a role in determining the outcome of T. cruzi infection (25, 40, 44). The main sources of the early production of these cytokines are macrophages and dendritic cells. While it is known that macrophage TNF- secretion is up-regulated by MIF (26), the role of MIF in regulating IL-1 and IL-18 remains elusive. To determine whether the production of proinflammatory cytokines is influenced by MIF during T. cruzi infection, we measured the levels of TNF-, IL-1, and IL-18 in the sera of MIF–/– and MIF+/+ mice during the course of infection. TNF- was detected in sera from both groups early during the course of infection, but the levels were significantly higher in MIF+/+ mice than in MIF–/– mice (Fig. 3A). However, as the infection progressed, both MIF+/+ and MIF–/– mice displayed comparable levels of TNF- in their sera. Similarly, TNF- levels in spleen and lymph node cell culture supernatants were also elevated in WT mice during the early phase of infection (data not shown). Interestingly, the kinetics of IL-1 and IL-18 production also displayed similar patterns, and serum levels of both of these cytokines were significantly higher in MIF+/+ mice than in MIF–/– mice throughout the course of the infection (Fig. 3B and C). These data suggest a pivotal role for MIF in regulating IL-1 and IL-18 production.
Augmented Th2-type reactions are not responsible for impaired proinflammatory responses in MIF–/– mice. Since Th2-type cytokines, such as IL-4 and IL-10, are involved in both susceptibility to T. cruzi infection and down-regulation of inflammatory cytokines (23, 30), we determined whether altered levels of these cytokines were responsible for the enhanced susceptibility of MIF–/– mice to acute T. cruzi infection. No significant differences were noted in the serum levels of IL-4 and IL-10 between T. cruzi-infected WT and MIF–/– mice at all time points examined throughout the course of the infection (Fig. 4A and B). The production of T. cruzi lysate-specific IL-4 by spleen cells from MIF–/– mice was lower early in the infection, whereas IL-10 production was similar between groups (Fig. 4C and D). Thus, it appears that elevated levels of Th2-type cytokines are unlikely to be responsible for the down-regulation of proinflammatory cytokine production observed in MIF–/– mice.
T. cruzi-infected MIF–/– mice develop severe heart and skeletal muscle pathologies. On day 22 p.i., the hearts and skeletal muscles of MIF+/+ mice showed low tissue parasitism with moderate inflammatory mononuclear cell infiltration but without major histopathological signs of lesions (Fig. 5A and C). In contrast, histopathology sections of infected MIF–/– mice showed an intense inflammatory reaction in the heart tissue (Fig. 5B and E), with a marked increase in amastigote nests (Fig. 5F) which was accompanied by severe heart injury due to large necrotic lesions. Damage was also more evident in the skeletal muscles of MIF–/– infected mice, which had large lesions and a larger number of amastigote nests than those of MIF+/+ T. cruzi-infected mice (Fig. 5C and D). Nevertheless, the heart was the more severely affected tissue, with larger numbers of amastigote nests and greater inflammatory infiltrates than the skeletal muscles of MIF–/– mice (Fig. 5E and F). These results suggest that MIF–/– mice succumb to the classical pathophysiology of the infection, which is typical of a severe acute phase of experimental Chagas' disease.
Analysis of IFN-, CCL5, IL-12 p35, IL12 p40, p19 (IL-23), and iNOS mRNA expression in infected hearts. Since MIF–/– mice displayed severe cardiac pathology, we examined the cytokine and chemokine expression in this organ. On day 22 after T. cruzi infection, total mRNA was isolated from hearts, and RT-PCR analyses were performed. As shown in Fig. 6A and B, infected hearts from MIF–/– mice displayed a significant increase in transcripts of IFN-, but similar levels for CCL5, compared to hearts from MIF+/+ mice. Furthermore, transcripts of IFN- were detectable in MIF–/– mice by using amounts of cDNA as low as 7 ng (Fig. 6A), while using this amount of cDNA yielded barely detectable IFN- transcripts from MIF+/+ mice. Interestingly, -actin transcripts were more intensely detected in MIF–/– T. cruzi-infected mice. Using 1 μg of mRNA, similar -actin intensities were detected for both groups (data not shown); however, when dilutions of heart mRNA were performed, it was evident that MIF–/– infected mice expressed -actin more intensely than MIF+/+ infected mice (Fig. 6A and B). This apparent lack of good quality control is explained by the known effects of intense T. cruzi infection on heart muscle cells, where the disruption of the actin cytoskeleton generates an increase in -actin production (39). This observation is consistent with the higher parasite burdens and severe pathology in the infected hearts of MIF–/– mice. In contrast, when another housekeeping mRNA was tested (GAPDH), no differences in the transcripts were detected between MIF–/– and MIF+/+ infected mice (Fig. 6A). Additionally, using real-time RT-PCR, we measured IL-12, IL-23, and iNOS mRNA levels that are likely involved in mediating the host defense against T. cruzi. As shown in Fig. 6C, T. cruzi-infected MIF+/+ mice showed significant increases in the levels of IL-12 p35, IL-12 p40, IL-23 p19, and iNOS mRNAs in their hearts compared to similarly infected MIF–/– mice.
IFN--dependent, antigen-specific IgG2a antibody titers are decreased in MIF–/– mice. IFN- and anti-T. cruzi specific antibodies have been shown to act synergistically in protecting mice against this parasite (31, 40). Given that IFN- is known to modulate IgG production, particularly that of IgG2a, and that IFN- levels were similar in late infections between MIF–/– and MIF+/+ mice, we decided to evaluate the kinetics of parasite-specific IgG1 and IgG2a antibodies during T. cruzi infection. Throughout the course of infection, T. cruzi-infected MIF–/– mice displayed lower titers of T. cruzi-specific IgG2a than did MIF+/+ mice, and the differences were statistically significant on days 21 and 28 p.i. (Fig. 7A), despite the fact that the levels of IFN- in both the sera and spleen cell supernatants were comparable between groups at these time points. In contrast, T. cruzi-specific IgG1 production was not severely affected by MIF deficiency (Fig. 7B).
DISCUSSION
Our study shows that MIF–/– mice are highly susceptible to experimental Chagas' disease caused by T. cruzi. Infection of MIF–/– mice with 5 x 103 blood trypomastigotes of the pathogenic T. cruzi strain (Queretaro) resulted in prolonged and high parasitemia levels, severe cardiac immunopathology, and increased mortality. MIF–/– mice also developed relatively high blood parasitemia levels and cardiac inflammation when infected with 1 x 103 metacylic promastigotes of the nonpathogenic strain of T. cruzi, but they eventually resolved the infection as efficiently as MIF+/+ mice. These findings indicate that MIF plays a role in the host defense against acute T. cruzi infection.
The proinflammatory cytokines IL-12, IFN-, and TNF- play an important role in mediating resistance to T. cruzi infection (24, 44, 59), and MIF promotes IL-12 and TNF- production in macrophages (11, 26). Furthermore, MIF also mediates host immunity against intracellular pathogens by activating macrophages and enhancing their microbicidal activity by increasing NO production (26). In the present study, we found that T. cruzi-infected MIF–/– mice produced significantly less IL-12, IL-18, IL-1, and TNF- during the early phase of infection. Furthermore, we also found that hearts from T. cruzi-infected MIF–/– mice contained significantly less iNOS mRNA than hearts from MIF+/+ mice. Taken together, these findings suggest that endogenous MIF is required for optimal production of proinflammatory cytokines and NO during the early phase of T. cruzi infection. Because MIF–/– mice produce significantly less NK-cell-activating IL-12 and IL-18, the lack of IFN- production early in infection in these mice can be explained by an inadequate activation of NK cells, which are by far the most important source of IFN- at early stages of T. cruzi infection (40). It is important that the numbers of NK cells and macrophages were not diminished in MIF–/– infected mice, as measured by flow cytometry (data not shown). However, the requirement of MIF for efficient production of IL-1, IL-18, and IL-12 in innate immunity against T. cruzi infection appears to be a new finding.
The major pathway reported for MIF-induced proinflammatory cytokine production has been attributed to its ability to regulate lipopolysaccharide responses through the modulation of TLR4 expression on macrophages (53). Interestingly, TLR4-mediated responses to T. cruzi have been reported as one of the main pathways for inducing early cytokine production associated with innate immunity (48). Thus, low expression of TLR4 may also be involved in the low inflammatory response observed in MIF–/– T. cruzi-infected mice.
It is known that at early stages of acute T. cruzi infection, cell infiltrates are necessary to control parasite replication; however, excessive infiltration can be dangerous for the host. In the present study, the hearts from T. cruzi-infected MIF–/– mice expressed low levels of proinflammatory cytokines, such as IL-12 and IL-23, and the enzyme iNOS, which is responsible for the production of nitric oxide, an effector molecule involved in killing of T. cruzi (21). IL-23 has recently been associated with the promotion of autoimmune diseases (32), but its role in infectious diseases is still unknown. To our knowledge, this is the first time that a lack of IL-23 expression has been associated with MIF deficiency. Nonetheless, we also found that MIF–/– mice develop intense cardiac inflammation despite the lack of strong proinflammatory cytokine production. This controversial observation can be explained by the fact that the infected hearts of MIF–/– and MIF+/+ mice displayed similar expression levels of CCL5, which facilitates the trafficking of leukocytes to the heart and favors the development of an inflammatory infiltrate. In fact, a recent study showed that mice lacking CCR5, a receptor for CCL5, develop high parasitemia levels and cardiac parasitism and show reduced survival following T. cruzi infection (19). In our study, we also found that infected hearts from MIF–/– mice contained significantly more IFN- than those from wild-type mice, which is likely to contribute to cardiac immunopathology. Nonetheless, MIF–/– mice were unable to clear the parasites from the hearts despite intense cardiac inflammation, suggesting that the cardiac damage may be due to the large numbers of amastigote nests instead of immunity-mediated damage caused by inflammation. Although the mechanism by which MIF contributes to protective host immunity against T. cruzi is not entirely clear, perhaps the activation of infiltrating cells is adversely affected in the absence of MIF signaling, resulting in poor effector function and reduced killing of parasites.
A previous study demonstrated that MIF plays a critical role in the activation of T cells (2). However, adaptive immunity appears not to be affected by MIF deficiency, since T. cruzi antigen-stimulated spleen cells from both MIF+/+ and MIF–/– mice proliferated similarly and produced comparable levels of Th1- and Th2-type cytokines 3 weeks after infection. Thus, impaired proinflammatory cytokine production cannot be attributed to excess production of IL-4 or IL-10, both of which are known to have anti-inflammatory effects and to participate in susceptibility to T. cruzi infection. These data are consistent with previous reports using MIF–/– mice for other parasitic models (51, 55). In fact, those studies demonstrated that the absence of MIF prevents the development of protective immunity against another intracellular parasite, Leishmania major, as well as the extracellular parasite Taenia crassiceps, by inhibiting macrophage microbicidal activity rather than preventing the development of Th1-like responses. Moreover, for both parasitic infections, higher levels of antigen-specific IFN- were detected in MIF–/– mice which failed to mediate host resistance. Thus, the finding that T. cruzi lysate-specific IFN- production was not impaired in MIF–/– mice late in infection was not unexpected. It is significant, however, that despite the similar levels of circulating IFN- and the increase in mRNA expression of IFN- in cardiac tissue, MIF–/– mice displayed a deficit in T. cruzi lysate-specific IgG2a production. Interestingly, a recent report using an asthma model with MIF–/– mice showed a similar finding, in which specific anti-OVA IgG2a antibodies were significantly reduced compared with those in MIF+/+ mice (42). In line with this, the absence of MIF could affect the ability of macrophages and B cells to respond to IFN-, and hence IFN- produced by MIF–/– mice may fail to induce the efficient macrophage activation and NO production required to eliminate T. cruzi. The reason for this lack of response remains unknown but can partially explain the lack of parasite clearance by MIF–/– mice.
The pathogenicity of T. cruzi strains has been associated with the expression of some virulence factors, such as trans-sialidases (50) and the molecule gp83, that parasites use to invade cells (62). Weakly invasive strains, such as Ninoa (20), either express low levels of these markers or do not express them at all. Thus, these strains are handicapped in the ability to rapidly invade target cells, and the host may have a longer opportunity to eliminate these parasites—through both innate and adaptive mechanisms, such as complement activation and specific antibodies—before they cause tissue damage. Hence, it is likely that a less intense immune response in MIF–/– mice is sufficient to control the nonpathogenic strains of T. cruzi.
In conclusion, our findings show that endogenous MIF is involved in host resistance against acute T. cruzi infection; the higher susceptibility of MIF–/– mice may involve mechanisms that impair innate cytokine production, suggesting that MIF participates in the activation of innate immunity to induce an early inflammatory cytokine response against T. cruzi infection.
ACKNOWLEDGMENTS
This work was supported by DGAPA-PAPIIT-UNAM grant IN212804 to M.R.S. and grant IN226402 to B.E. and by an NIH grant to A.R.S.
We thank Ignacio Martínez, Benito S. Sánchez, and Rodrigo Velázquez for their technical assistance.
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