Toxoplasma gondii Infection Reveals a Novel Regulatory Role for Galectin- in the Interface of Innate and Adaptive Immunity

【摘要】  In attempts to investigate the role of galectin-3 in innate immunity, we studied galectin-3-deficient (gal3C/C) mice with regard to their response to Toxoplasma gondii infection, which is characterized by inflammation in affected organs, Th-1-polarized immune response, and accumulation of cysts in the central nervous system. In wild-type (gal3+/+) mice, infected orally, galectin-3 was highly expressed in the leukocytes infiltrating the intestines, liver, lungs, and brain. Compared with gal3+/+, infected gal3C/C mice developed reduced inflammatory response in all of these organs but the lungs. Brain of gal3C/C mice displayed a significantly reduced number of infiltrating monocytes/macrophages and CD8+ cells and a higher parasite burden. Furthermore, gal3C/C mice mounted a higher Th1-polarized response and had comparable survival rates on peroral T. gondii infection, even though they were more susceptible to intraperitoneal infection. Interestingly, splenic cells and purified CD11c+ dendritic cells from gal3C/C mice produced higher amounts of interleukin-12 than cells from gal3+/+ mice, possibly explaining the higher Th1 response verified in the gal3C/C mice. We conclude that galectin-3 exerts an important role in innate immunity, including not only a pro-inflammatory effect but also a regulatory role on dendritic cells, capable of interfering in the adaptive immune response.
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Galectins are a family of animal lectins composed of 15 members that are conserved throughout animal evolution.1-4 They recognize galactose-containing oligosaccharides and share sequence similarities in their carbohydrate-recognition domain. Several immune cells differentially express galectins, and their expression levels appear to be dependent on cell differentiation and activation. They can interact with cell surface glycoconjugates decorated with suitable saccharides and then trigger cell growth and migration, as well as modulation of cell survival.5 They can also modulate cellular activities by functioning intracellularly.6
In the past few years, the concept has emerged that some members of the galectin family might play an essential role in the initiation and amplification of the inflammatory response, whereas other members exert a suppressive role in the inflammatory response.7 Thus, in contrast to the anti-inflammatory effect of galectin-1, a powerful pro-inflammatory activity has been proposed for galectin-3. Galectin-3 consists of an N-terminal domain (about 120 amino acids) made of tandem repeats of short stretches of amino acids connected to a C-terminal carbohydrate-recognition domain.8 Various extracellular and intracellular activities of galectin-3 have so far been demonstrated in vitro. When intracellularly localized, galectin-3 has been appointed as an anti-apoptotic molecule9,10 and implicated in diverse biological processes, including cell differentiation11 and pre-mRNA splicing induction.12 On the other hand, extracellular galectin-3 can function as an inducer of T-cell apoptosis through binding to CD7 and CD29 on the T-cell surface,13 as well as a positive or a negative growth factor, depending on the target cells.14,15 Extracellular galectin-3 can also promote cell adhesion, including that of human neutrophils to laminin16 and of L-selectin-activated lymphocytes to dendritic cells.17 In addition, it can activate some cell types, such as mast cells,18 neutrophils,19,20 and monocytes.21
Studies of galectin-3-deficient (gal3C/C) mice22,23 have provided significant functional insights of this protein. These animals do not exhibit any developmental or morphological abnormalities, and young adult mice have so far presented no apparent phenotypes under standard laboratory conditions. However, several studies have provided significant support for the pro-inflammatory22-24 and phagocytotic25 roles of galectin-3 and for its function as a receptor of "advanced glycation end" products.26 Nevertheless, despite considerable progress in elucidating galectin-3 functions, the mechanisms underlying the immunomodulatory properties of this lectin remain to be elucidated. Thus, we have used experimental Toxoplasma gondii infection to investigate the immunoregulatory properties of galectin-3, taking advantage of the availability of gal3C/C mice.
T. gondii is an intracellular parasite that influences host resistance by affecting functions in various immune cell types. The disease is generally initiated by an acute phase, associated with rapid tachyzoite proliferation, followed by a chronic stage, mainly characterized by the presence of latent cysts within the central nervous system and skeletal muscles.27 Studies using toxoplasmosis mouse models have clearly demonstrated that resistance is associated with the activation of a strong cell-mediated Th1-type immune response, which is associated with high interferon (IFN)- production driven by interleukin (IL)-12 derived from dendritic cells.27-29 To determine whether galectin-3 could participate in this immunoregulation, we have analyzed its role in the initial events responsible for the induction and regulation of the immune response against the parasite.
In the present study, we demonstrate that the absence of galectin-3 increases IL-12 production by dendritic cells, driving the development of a heightened Th-1-type immune response. Because IL-12 is a cytokine that bridges innate and adaptive immunity, we hypothesize that galectin-3 may play a role in tuning up both the innate and adaptive responses to different pathogens.

【关键词】  toxoplasma infection regulatory galectin- interface adaptive immunity

Materials and Methods

Experimental Animals

The galectin-3-deficient (gal3C/C) mice were generated as previously described23 and backcrossed to C57BL/6 mice for nine generations. Age-matched wild-type (gal3+/+) mice in a C57BL/6 background were used as control in all of the experiments. Mice were housed under approved conditions at the Animal Research Facilities of Faculdade de Medicina de Ribeirão Preto-USP. All of the animals used in the experiments were 6- to 8-week-old males.

Parasites and Antigen Preparation

The low-virulent ME-49 strain of T. gondii was used to infect the mice.30 Cysts were harvested from the brains of C57BL/6 mice that had been inoculated with approximately 10 cysts through the intraperitoneal route 1 month before. For preparation of the T. gondii tachyzoite-soluble antigen (STAg), tachyzoites of the RH strain were harvested from the peritoneal cavities of out-bred Swiss Webster mice that had been injected with 107 organisms 2 days earlier. The tachyzoites were sonicated and centrifuged, and the supernatant was collected as previously described.31 The Ethics Committee on Animal Research of the University of Sao Paulo approved all procedures performed in the studies described herein.

Infection of Mice and Tissue Processing

Gal3+/+ and gal3C/C mice were perorally infected with 20 T. gondii cysts (groups of four mice each). On days 7, 14, 21, and 28 after infection, the mice were euthanized; samples of the brain, lung, liver, spleen, kidney, and small intestine tissues were collected, fixed in 10% buffered formalin, and processed routinely for paraffin embedding and sectioning. The small intestine was cut into four pieces, and each piece was rolled on itself to make a "Swiss roll." The entire length of the small intestine was examined histologically. For each organ, tissue sections of 4 µm thickness (40-µm distance between sections) were mounted on slides for histopathological and immunohistochemical studies. These sections were stained with hematoxylin and eosin (H&E) and observed under an optical microscope. gal3+/+ and gal3C/C mice were also infected intraperitoneally with 20 T. gondii cysts. On days 1 and 4 after infection, the mice were euthanized, and peritoneal cells were harvested by injection of 5 ml of ice-cold phosphate-buffered saline (PBS) supplemented with 10 mmol/L HEPES and 0.1 mmol/L EGTA. For the recovery of cellular contents, recovered lavage fluid was centrifuged at 200 x g for 10 minutes at 4??C. The total cell counts were determined using diluting fluid in a Neubauer chamber, and differential counts were performed using Rosenfeld-stained cytospin preparations. On day 14 after intraperitoneal infection, cyst numbers were counted from whole brain homogenates.

Immunohistochemistry Studies

To detect galectin-3 expression by immunohistochemistry, deparaffinized sections were incubated for 1 hour in 2% normal goat serum/1% bovine serum albumin (BSA) to reduce nonspecific binding. The slides were then incubated with rat anti-mouse galectin-3 monoclonal antibody (M3/38)32 diluted in 1% BSA for 1 hour. The secondary antibodies labeled with peroxidase were goat anti-rat IgG antibodies (Sigma Chemical Co., St. Louis, MO). The reaction was visualized by incubating the section with 3,3'-diaminobenzidine tetrahydrochloride (Amresco, Solon, OH) for 5 minutes. In the control slides, normal rat IgG replaced the primary antibody. The slides were studied with an Olympus microscope (Olympus Co., Tokyo, Japan) and photographed with Kodak film (100 ASA, Rochester, NY). All steps were performed at room temperature. Samples of the brain were cryopreserved for the study of the presence of CD4+, CD8+, and F4/80+ cells. Serial 5-µm-thick sections were prepared, fixed in cold acetone, and stained with immunoperoxidase as previously described.33 Immunoperoxidase staining with polyclonal rabbit antibodies against T. gondii was also used for detection of the parasites.34

Histological Analysis

The tissue parasitism detected by immunohistochemistry was scored by counting the total number of cyst-like structures and parasitophorous vacuoles in 40 microscopic fields (x400) per histopathological section (40-µm distance between sections) as described previously.35 For the inflammatory infiltrate score, the total numbers of focal or diffuse inflammatory foci were counted in 25 microscope fields (x200) per tissue section. For both tissue parasitism and inflammation scores, the quantifications were performed in four noncontiguous sections (40-µm distance between them) in a blinded manner by two researchers.35,36 Four sections were counted for each animal, and individual data points were determined from the mean result of the four sections.

Spleen Cell Cultures

The ME-49 T. gondii-infected gal3+/+ and gal3C/C mice were euthanized 7, 14, 21, and 28 days after infection, and their spleens were removed. The suspensions of spleen cells were washed in RPMI and treated for 2 minutes with lysing buffer (9 volumes of 0.16 mol/L NH4Cl and 1 volume of 0.17 mol/L Tris-HCl, pH 7.5). The erythrocyte-free cells were then washed three times and adjusted to 2 x 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. The cell suspension was distributed (1.0 ml/well) in a 24-well tissue culture plate at 37??C in 5% CO2, and STAg (5 µg/ml) was added to each well. Spleen cells from uninfected mice were also used and treated with lipopolysaccharide (LPS; 5 µg/ml; Sigma), IFN- (10 ng/ml; BD PharMingen, San Diego, CA), or LPS + IFN-. The culture supernatants were collected after 48 hours for determination of cytokine levels, as described below.

Purification of Splenic CD11c+ Dendritic Cells and Macrophages from Uninfected gal3+/+ and gal3C/C Mice

Dendritic cells (DCs) were purified from the spleen as previously described,37 with modifications. Briefly, spleens were digested for 30 minutes at 37??C with 1 ml of 1 mg/ml collagenase type V (Sigma) solution and divided into low- and high-density fractions on a dense BSA solution (Sigma). Further purification of fresh dendritic cells by enrichment on a Mini MACS column was performed according to the manufacturer??s recommendations (Miltenyi Biotec, Auburn, CA). This procedure yielded 60 to 80% pure dendritic cell populations.

To isolate splenic macrophages, spleen cells were seeded into tissue culture-treated plastic petri dishes (Falcon 3001; VWR, Edmonton, Canada) and incubated at 37??C in a 5% CO2 incubator. After 3 hours, the nonadherent cells were removed by pipetting and careful washing, and the adherent cells were dislodged with ice-cold versene (0.02% ethylenediamine tetraacetic acid in PBS, pH 7.2) and gentle scraping. All cells were washed with complete medium, and their purity was determined by Giemsa staining. On all occasions, the adherent cells were mostly macrophages (85%), as previously demonstrated.38

Induction of IL-12 Production by Splenic Macrophages and CD11c+ Dendritic Cells

Splenic dendritic cells and macrophages from uninfected mice were both isolated as described above. Splenic DCs (2 x 105/well) and adherent macrophages (2 x 105/well) were cultured and stimulated for 48 hours with 5 µg/ml LPS (Sigma), 10 ng/ml IFN- (PharMingen), or LPS (5 µg/ml) plus IFN- (10 ng/ml). After incubation, the culture supernatants were collected, separated from cells by centrifugation, and stored at C70??C until analysis. The resulting IL-12p40 production was measured by enzyme-linked immunosorbent assay (ELISA), as described below.

Cytokine Measurement

IL-12p40, IL-12p70, IFN-, IL-4, and IL-10 present in serum and total spleen cell supernatant were quantified by ELISA with a commercially available kit, according to the manufacturer??s instructions (OptEIA set; PharMingen). The sensitivity limits of the assays were 15 pg/ml for IL-12p40 and IL-12p70, 7 pg/ml for IL-4, and 30 pg/ml for IFN- and IL-10.

Quantification of Parasite-Specific IgG1 and IgG2a

For the determination of Ig production, levels of antigen-specific IgG1 and IgG2a antibodies in the serum samples were determined by ELISA. Microtiter plates (Nunc, Naperville, IL) were coated with 10 µg/ml T. gondii antigen diluted in carbonate-bicarbonate buffer (0.1 mol/L, pH 9.6) overnight at 4??C. The plates were then washed three times in PBS containing 0.05% Tween 20 (PBS-T) (pH 7.4). Nonspecific binding sites were blocked with PBS-T containing 1% BSA (blocking buffer) for 1 hour at 37??C. Serum samples (50 µl) were added to duplicate wells at a 1/10 dilution in blocking buffer. Plates were then incubated at 37??C for 1 hour, washed four times, and incubated with peroxidase-conjugated goat anti-mouse IgG1 or IgG2a antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1/10,000 dilution in blocking buffer for 1 hour at 37??C. After washing with PBS-T, reactions were developed with the TMB (3,3',5,5' tetramethylbenzidine) Substrate Kit according to the manufacturer??s instructions (Pierce Chemical Co., Rockford, IL). The reaction was stopped 15 minutes later by addition of 1 mol/L sulfuric acid to each well. The absorbance was read at 450 nm in a Microplate Scanning Spectrophotometer (PowerWavex; Bio-Tek Instruments, Inc., Winooski, VT). Antibody titers were arbitrarily expressed as ELISA Indexes (EI), according to modifications to the method of Turunen et al,39 as follows: EI = (ODsample/ODcutt-off), where values of EI 1.0 were considered as cut-off titers.

Statistical Analysis

Statistical analysis of control and experimental groups was accomplished by Student??s t-test using the GraphPad InStat 3 software. A P value less than 0.05 was considered statistically significant. Finally, the log rank test and Kaplan-Mayer curves were used to compare the survival rates between the study groups, and differences were considered statistically significant when P < 0.05.

Results

Galectin-3 Is Up-Regulated in Various Tissues in Response to T. gondii Infection

We investigated whether galectin-3 expression was up-regulated in response to T. gondii infection. For this purpose, the peripheral organs (intestine, liver, spleen, and lungs) and the brain from orally infected mice were evaluated by immunohistochemistry for galectin-3 on days 7, 14, 21, and 28 after infection, and results were compared with those obtained for the uninfected mice. Regarding the peripheral organs, galectin-3 was detected at low levels in tissues from the uninfected mice (Figure 1, A and B) . However, by day 7 after infection, the staining substantially increased (data not shown) and remained high until day 14, coinciding with the high inflammatory infiltration in the organs (Figure 1, D and E) . The increased staining was localized on the infiltrating cells. On day 28, when the parasite could not be detected in the peripheral tissues, galectin-3 staining decreased, reaching levels close to those observed in the uninfected mice (Figure 1, G and H) .

Figure 1. Immunohistochemical staining for galectin-3 in organs of T. gondii-infected wild-type mice. The photomicrographs depict galectin-3 immunostaining (brown color) with a hematoxylin counterstain. In the uninfected mice, few cells in the liver (A) and spleen (B) express galectin-3, and galectin-3 staining is barely noted in the brain (C). On day 14 after T. gondii infection, galectin-3 is expressed by a high number of inflammatory cells in the liver (D), spleen (E), and brain (F). On day 28 after T. gondii infection, the level of galectin-3 expression in the liver (G) and spleen (H) decline to almost basal levels but remain high in the brain (I). All photomicrographs are under magnification with x40 objective. Circles show inflammatory foci, and arrows point to galectin-3-expressing cells.

Galectin-3 staining was absent in the brain from uninfected mice (Figure 1C) . However, high galectin-3 staining was detected on days 14, 21, and 28 after infection (Figure 1, F and I , for days 14 and 28, respectively). The increase in galectin-3 staining coincided with the presence of the parasites in the brain and was localized in the infiltrating cells. Also, there was an increase in galectin-3 staining in the inflamed lungs when compared with the normal lungs (data not shown). As expected, no appreciable staining was noted when tissue sections from the gal3C/C mice were stained by the same anti-galectin-3 antibody (data not shown).

Gal3C/C Mice Have a Milder Inflammatory Response but a Similar Parasite Burden in the Intestine when Compared with gal3+/+ Mice

Because oral infection by T. gondii is typically followed by proliferation of parasites in the small intestine40 and because susceptibility of C57BL/6 mice to oral infection is partially attributed to necrosis of the small intestinal villi,41 we compared tissue parasitism and inflammatory response in the small intestine of the infected mice. In both gal3+/+ and gal3C/C mice, lesions of the small intestine were more clearly detectable from days 6 to 8 after infection. Histological analysis of the small intestine from gal3+/+ mice on day 6 revealed increasing thickness of the villi and leukocyte infiltration into lamina propria of the ilea (Figure 2A) . In contrast, only mild leukocyte infiltration was observed in gal3C/C mice (Figure 2B) . In addition, necrosis of the villi was noted in gal3+/+ mice 6 days after infection (Figure 2A) , whereas no such lesions were detected in gal3C/C mice (Figure 2B) . On day 8 after infection, large necrotic areas were observed in the ilea of the gal3+/+ mice, predominantly within the villi, and some villi were completely destroyed (data not shown). The inflammatory reaction progressively decreased in both gal3+/+ and gal3C/C mice after 8 days of infection (Figure 3A) . Despite the marked difference in the inflammatory response, there was no significant difference in the parasite burden in both gal3+/+ and gal3C/C mice (52 ?? 28.9 and 37 ?? 26.4, respectively).

Figure 2. Histopathology and inflammatory response in tissues of T. gondii-infected gal3+/+ and gal3C/C mice. Mice were infected with 20 cysts of the ME-49 strain of T. gondii, and histological studies were performed on their intestine, liver, and lung at several intervals after infection. A and B: The intestines of gal3+/+ and gal3C/C mice, respectively, show initial stages of tissue necrosis accompanied by an increased number of inflammatory cells in the lamina propria and submucosa on day 6 (A). In contrast, gal3C/C mice exhibited a preserved villous architecture on day 6 (B). C and D: The liver of gal3+/+ and gal3C/C mice, respectively, on day 14 after infection. E and F: The lung of gal3+/+ and gal3C/C mice, respectively, on day 28 after infection. Stronger inflammatory infiltration persists in the lung of gal3C/C. H&E; A, B, E, and F, under magnification with x10 objective; C and D, under magnification with x60 objective.

Figure 3. Summary of the inflammatory response kinetics in the peripheral organs and CNS of T. gondii-infected gal3+/+ and gal3C/C mice. A (intestine), B (liver), C (lung), and D (CNS) show the evolution of inflammation intensity at several intervals after infection. The inflammatory scores were measured as described in Materials and Methods. Asterisks indicate that differences are statistically significant at P < 0.05.

Gal3C/C Mice Have a Lower Inflammatory Response in the Liver but a Higher Inflammatory Response in the Lungs when Compared with gal3+/+ Mice

It is known that the parasite disseminates through the blood vessels to other organs after reaching the small intestine.40 We thus compared the inflammatory reaction in the liver and lungs of the gal3+/+ and gal3C/C mice. As summarized in Figure 3B , the inflammatory reaction in the liver was less intense in gal3C/C mice, especially on day 14. Representative photomicrographs of hepatic lesions found in gal3+/+ and gal3C/C mice are shown in Figure 2, C and D . A different picture was noted regarding the lungs, where a similar inflammatory infiltration was detected in the two genotypes during the first 2 weeks of infection (Figure 3C) . In the following 2 weeks, however, the inflammation progressively decreased in gal3+/+ mice (Figure 2E) but persisted in gal3C/C mice (Figure 2F) . Thus, on day 21, there were more inflammatory infiltrates in gal3C/C mice when compared with gal3+/+ mice (Figure 3C) . Considering the highly persistent inflammatory infiltration observed in the lungs of gal3C/C mice, we also measured the pulmonary parasite burden. We found that the number of parasitophorous vacuoles was dramatically higher in gal3C/C compared with gal3+/+ mice (20.3 ?? 4.8 versus 2.5 ?? 0.7, respectively) on day 7 after infection. The tissue parasitism was still higher in gal3C/C than in gal3+/+ mice on day 14 (22.5 ?? 3.2 versus 2.25 ?? 0.5) but sharply declined in both the gal3C/C and gal3+/+ mice on day 21 after infection (5.7 ?? 1.6 versus 0.7 ?? 0.6, respectively). On day 28, virtually no parasitophorous vacuoles were detected in both groups. This result indicates that the persistently high inflammatory reaction in the lungs of the gal3C/C mice does not depend only on the parasite burden.

The Central Nervous System of gal3C/C Mice Exhibits a Higher Parasite Burden and Delayed Inflammatory Response

Around 7 to 9 days after infection, tachyzoites reach the brain42 where they are converted into bradyzoites, culminating with the formation of cysts that remain for the whole life of the host. We thus investigated the inflammatory response and parasitism in the brain. As shown in Figure 3D , the inflammation exhibited by gal3C/C mice during the first 3 weeks of infection was lower than that displayed by the gal3+/+ mice. However, by day 28 after infection, the inflammation exhibited in the two genotypes was comparable.

Analysis on day 7 after infection revealed the presence of few free parasites in the brain of both gal3+/+ and gal3C/C mice. Remarkably, 14 days after infection, the number of cysts in gal3C/C mice was 11 times higher than that in gal3+/+ mice (Figure 4, A and B) . There were more cysts in gal3C/C mice throughout the experimental period, although the ratio between the numbers of cysts detected in gal3C/C versus gal3+/+ mice decreased to 6 on day 21 and to 3 on day 28 (Figure 4B) . To determine the subsets of infiltrating mononuclear cells in the brain of T. gondii-infected mice, we counted the numbers of CD4+, CD8+, and F4/80+ cells on days 7, 14, and 28 by immunohistochemistry. Although no differences were found on day 7, gal3C/C mice showed a striking reduction in the number of infiltrating F4/80+ macrophages on days 14 and 28 and in the number of CD8+ cells on day 28 (Figure 4, C and D) . The number of CD4+ cells did not differ significantly between the two genotypes during all intervals analyzed. These results suggest that the higher parasite replication in perorally infected gal3C/C mice may be related to the lower levels of infiltrating macrophages and CD8+ cytotoxic T lymphocytes in the brain.

Figure 4. Parasite burden in the brain of T. gondii-infected gal3+/+ and gal3C/C mice. The mice were perorally infected with 20 cysts of the ME-49 strain of T. gondii. A: Arrows and ellipse indicate cysts in the brains of gal3+/+ and gal3C/C mice on day 14 after infection (parasite-specific immunostaining; magnification with x40 objective). B: The number of cysts detected by immunocytochemistry in brain sections of T. gondii-infected gal3+/+ and gal3C/C mice during the course of the infection. *Significantly different from values obtained with the gal3+/+ mice (P < 0.03). Similar results were obtained in two experiments. C and D: The numbers of infiltrating CD4+, CD8+, and F4/80+ cells into the brains of T. gondii-infected gal3+/+ and gal3C/C mice 14 and 28 days after infection, respectively. Cells were counted by immunohistochemistry, and data represent the mean ?? SEM, *P < 0.05.

The Mortality of gal3C/C Mice Is Comparable with That of Their Wild-Type Counterpart when Orally Infected with T. gondii, but It Is Higher when Intraperitoneally Infected

We next compared the outcome of the T. gondii infection in gal3+/+ and gal3C/C mice, both in the C57BL/6 background, which is known to be susceptible to T. gondii infection.43 For this purpose, mice inoculated perorally with 20 cysts of the ME-49 strain of T. gondii were monitored for survival. Both gal3+/+ and gal3C/C mice survived for at least 30 days after infection but started dying thereafter (Figure 5A) . There was no difference in survival out to 100 days between the two genotypes. However, gal3C/C mice exhibited significantly lower survival rates than gal3+/+ mice when infected intraperitoneally (P < 0.027) (Figure 5B) . To determine the mechanism involved in the lower survival of the intraperitoneally infected gal3C/C mice, we investigated the inflammatory infiltration into the peritoneal cavity and the parasite burden during the early stages of infection. On day 4 after infection (intraperitoneal) with 20 ME-49 cysts, infiltration of neutrophils (Figure 6A) and monocytes/macrophages (Figure 6B) into the peritoneal cavity was respectively lower by 47 and 61% in gal3C/C mice. In addition, on day 14 after infection, the number of cysts found in the brain of gal3C/C mice was five times higher than that in gal3+/+ mice (Figure 6C) . Thus, we suggest that deficient leukocyte recruitment during the early stages of intraperitoneal infection contributed to a higher parasite burden and decreased survival of gal3C/C mice. Moreover, depending on the route of infection, galectin-3 contributes to the overall susceptibility of mice to T. gondii infection.

Figure 5. Survival of gal3+/+ and gal3C/C mice to T. gondii infection. Mice were perorally (A) or intraperitoneally (B) infected with 20 cysts of the ME-49 strain of T. gondii, and survival was monitored. Data are representative of three experiments, each performed with five to eight mice per group, yielding similar results. The results are expressed as percentage of live animals during the course of infection. A: No difference in mortality was observed between gal3+/+ and gal3C/C after oral infection. B: The gal3C/C mice infected by the intraperitoneal route exhibit significantly lower survival than gal3+/+ mice infected by the same route (P < 0.027).

Figure 6. Reduced recruitment of inflammatory monocytes/macrophages and neutrophils, and high parasite burden in gal3C/C mice after intraperitoneal infection with T. gondii. Peritoneal cells from gal3+/+ and gal3C/C mice were harvested 4 days after intraperitoneal inoculation of a dose of 20 cysts of the ME-49 strain of T. gondii, and subpopulations of leukocytes in the fluid were enumerated. A shows the recoveries for neutrophils and B for macrophages 4 days after infection (pi). C: Shows cyst numbers counted from whole brain homogenates 14 days after infection (pi). Results shown are representative of individual mice. The experiment was repeated twice with similar results, using a total of five mice per genotype. *P < 0.03.

Gal3C/C Mice Mount a Higher Th1 Immune Response when Compared with gal3+/+ Mice

It is known that T. gondii induces a strong IL-12-dependent cell-mediated immune response that restrains the replication of tachyzoites. It is also a common knowledge that resistance to parasites is associated with an IFN--dependent response triggered by IL-12.27 We thus assessed the concentrations of Th1 cytokine and IgG isotypes in the serum. Interestingly, the concentrations of IL-12p40 and IFN- were significantly higher in the sera of gal3C/C mice compared with those of gal3+/+ mice (Figure 7, A and B) . To corroborate the finding of the enhanced Th1-type immune response in gal3C/C mice, we determined the ratio of IgG2a to IgG1 antibodies specific for T. gondii antigens in the sera from infected gal3+/+ and gal3C/C mice. Consistent with a heightened Th1 response, gal3C/C mice exhibited a higher IgG2a-to-IgG1 ratio (Figure 7C) .

Figure 7. Levels of IL-12p40 and IFN- and ratio of parasite-specific IgG2a to IgG1-isotype in the sera of T. gondii-infected mice. The serum samples were collected on day 14 after infection. IL-12p40 (A), IFN- (B), and IgG2a/IgG1 (C) levels were assayed by ELISA. The results represent the mean ?? SD of data from five mice of each genotype. *P < 0.03. A: The difference between the responses obtained with the gal3+/+ and gal3C/C mice are significantly different throughout the entire period (P < 0.05). Similar results were obtained in three experiments.

We also cultured spleen cells from the infected mice and assayed them for cytokine production. The gal3C/C cells were found to release higher amounts of IL-12p40 at all time points, even in the absence of stimulation (Figure 8A) . In addition, the gal3C/C cells released higher levels of IFN- up to day 14 after infection (Figure 8B) ; the levels of this cytokine were below the limit of detection in both genotypes thereafter. In contrast, the two genotypes did not differ in the levels of IL-10 (data not shown), whereas the levels of IL-4 were under the detection limit.

Figure 8. Levels of IL-12p40 in the supernatants of spleen cell from T. gondii-infected mice. Spleen cells, harvested on days 7, 14, 21, and 28 after infection, were incubated with medium alone for 48 hours. The supernatants were collected and assayed for IL-12p40 (A) and IFN- by ELISA. A: The results represent the mean ?? SD of triplicate cultures from a representative experiment. *P < 0.02. The difference between the responses obtained with the gal3+/+ and gal3C/C mice are significantly different throughout the entire period (P < 0.05). B: Levels of IFN- in the supernatant of the spleen cells harvested on days 7 and 14 after infection, without any re-stimulation. After day 15, IFN- in the supernatant is below the detection limit in all groups, unless STAg stimulation is used. Results are reported as the mean ?? SD. *P < 0.05. The experiment was repeated a second time with similar results.

High IL-12 Production by Cells of Uninfected gal3C/C Mice: the Role of Dendritic Cells

We investigated whether cells of uninfected gal3C/C mice also produced higher levels of Th1 cytokines. Increased levels of IFN- (Figure 9A) and IL-12p40 (Figure 9B) were produced by spleen cells of gal3C/C mice, on in vitro stimulation with LPS and IFN-. Because the induction of the p40 subunit often results in production of IL-12 active form,44 we assessed the IL-12p70 levels in spleen cell supernatants after stimulation with LPS and IFN-. Cells from gal3C/C mice also produced higher levels of IL-12p70 (Figure 9C) when compared with gal3+/+ mice. Thus, considering that DCs are the cells that first release IL-12 after in vivo stimulation with T. gondii antigens or LPS,29 we sought to investigate whether this cell type accounts for the increased levels of IL-12p40 found in gal3C/C mice. Therefore, we purified CD11c+ splenic DCs of uninfected gal3C/C and gal3+/+ mice and assayed the cytokine production 48 hours after stimulation with LPS and IFN-. As shown in Figure 9D , CD11c+ DCs, but not macrophages, account for the higher levels of IL-12p40 found in the spleen cell supernatants of gal3C/C mice. These results imply that, in the absence of galectin-3, increased IL-12 production by dendritic cells is at least partially responsible for the higher Th1 response developed by gal3C/C mice.

Figure 9. Production of IL-12p40, IL12p70, and IFN- by the spleen cells from uninfected gal3+/+ and gal3C/C mice. Total cells, macrophages, and CD11c+ dendritic cells isolated from the spleen of uninfected gal3+/+ and gal3C/C mice were cultured for 48 hours in the presence or absence of LPS, IFN-, or LPS + IFN-. The concentrations of IFN- (A), IL-12p40 (B), and IL-12p70 (C) in the culture supernatants were measured by ELISA. The spleen cells of gal3C/C mice produced higher levels of IFN- (A), IL-12p40 (B), and IL-12p70 (C) when compared with those of gal+/+ mice. CD11c+ dendritic cells but not macrophages account for the higher levels of IL-12p40 in gal3C/C mice (D) after stimulation with LPS + IFN-. The results represent the mean ?? SD of triplicate cultures from a representative experiment. *P < 0.02.

Discussion

The major findings resulting from T. gondii murine infection are that compared with wild-type mice, gal3C/C mice 1) develop reduced inflammatory responses in all organs, except for the lungs; 2) exhibit a higher parasite burden in the lungs and the brain; 3) mount a higher Th1-polarized response, manifested by higher levels of IL-12 and IFN- and a higher ratio of T. gondii-specific IgG2a to IgG1 in the sera; and 4) exhibit an up-regulation of IL-12 production by dendritic cells. Despite these differences, gal3C/C mice and gal3+/+ mice are comparable in their survival rates on peroral T. gondii infection. In contrast, when challenged by the intraperitoneal route, gal3C/C mice show decreased survival rates. Although the delayed inflammatory response seen in the gal3C/C mice may result in higher parasite spreading, an overproduction of IL-12 and the consequent higher levels of IFN- led to the development of a heightened Th1 response needed for controlling the parasite infection. Although we did not exclude its role on other cell types, we postulate that galectin-3 plays a regulatory role in immunity through novel mechanisms: it regulates the immune response at least in part by influencing IL-12 production by DCs, thus exerting a profound effect on the development of the adaptive immune response against pathogens. The results reported in this study corroborate the pro-inflammatory role of galectin-3 previously reported and provide evidence that galectin-3 is a tuner of the immune system, being able to adjust the intensity of the immune response by regulating the innate response and the development of the adaptive response. Previous studies of peritonitis induction in gal3C/C mice have provided significant support for the pro-inflammatory role of this lectin. After injection of a noninfectious inflammatory agent, thioglycollate broth, gal3C/C mice exhibited a significantly reduced number of granulocytes and macrophages in the peritoneal cavity.22,23 However, the absence of galectin-3 has been reported to affect the migration of macrophages, eosinophils, neutrophils, and lymphocytes in a different way depending on the experimental conditions.22-25 It is difficult to address the exact mechanism behind its pro-inflammatory role. In our experimental model, gal3C/C mice exhibited a deficient inflammatory response in the small intestine, liver, and central nervous system during acute infection. However, the inflammatory reaction in the lungs from gal3C/C mice was similar to that seen in the gal3+/+ mice at the early phase but was stronger in the late phase of infection. These results indicate that other mechanisms are involved in the emigration of inflammatory cells into the lung and/or that galectin-3 exerts a different role on cellular migration depending on the examined organ. Considering that the parasite was already eliminated from the lung on day 28 after infection (data not shown) and knowing that leukocyte migration in the lung has very particular features when compared with other peripheral organs,45 further studies should be performed to elucidate the mechanism of such late hyperresponsiveness.

The finding that the mortality rate between gal3+/+ and gal3C/C mice was comparable when the mice were infected with the parasites through the oral route is likely to be the result of the variable inflammatory responses in different organs in these two strains of mice. The mortality rate in T. gondii infection is thought to be associated with tissue inflammation.

When challenged with 100 ME-49 strain tissue cysts, it has been reported that female C57BL/6 mice are mainly susceptible because of a strong inflammatory response developed against the parasite in the small intestine, which causes severe local necrosis.41 Mice deficient in the anti-inflammatory cytokine IL-10 succumbed during early infection by T. gondii as a consequence of the increased frequency and intensity of cellular infiltration and tissue necrosis, rather than because of uncontrolled parasite growth.46 Additional support is derived from studies with mice that are unable to produce nitric oxide. On challenge with T. gondii, mice deficient in nitric oxide synthase exhibited prolonged survival and fewer pathological changes in their liver and gut, despite presenting an uncontrolled dissemination of the parasite.47 Thus, the milder inflammation in gal3C/C mice in the intestine would account for the lesser extent of tissue damage and favor their survival. On the other hand, the reduced number of infiltrating macrophages and CD8+ cells in the brain of gal3C/C mice most likely contributed to the higher burden of tissue cysts found. Although the CD4+ subset may be important for mounting and sustaining the T-cell response against T. gondii, it is the CD8+ subset that appears critical for host survival.48,49 It has been demonstrated that mice vaccinated with the ts-4 mutant, allowing development of a strong protective immune response against subsequent challenge with the virulent RH strain, become more susceptible to T. gondii infection after CD8+ cell depletion, achieved by using monoclonal antibodies.31,49 Interestingly, the higher mortality observed in gal3C/C mice after intraperitoneal infection with T. gondii was associated with a deficient influx of neutrophils and macrophages to the peritoneal cavity on day 4 after infection. It has been shown that CCR2C/C mice fail to recruit monocytes/macrophages to the peritoneal cavity during the early stages of intraperitoneal infection with T. gondii, resulting in greatly enhanced mortality despite the induction of normal Th1 cell responses.50 Thus, we hypothesized that the galectin-3-dependent recruitment of macrophages and CD8+ cells may be an important mechanism of resistance in toxoplasmosis.

Several reports have indicated that galectin-3 may play a role in inflammation, but to date, no one has clearly demonstrated how galectin-3 can tune up both the innate and adaptive immune responses. The idea that galectin-3 can interfere with the adaptive response has so far been based on 1) the fact that the inhibition of galectin-3 by an antisense strategy blocked proliferation of T-cell receptor (TCR) TCR-stimulated T lymphocytes51 ; 2) its anti-apoptotic role, interfering in T-cell homeostasis and function9 ; 3) its potential role in allergic inflammation, specifically inhibiting the Th2-type cytokine IL-5 in human eosinophils52 ; and 4) its role in promoting dendritic cell/naïve T lymphocyte interaction.17 However, the strongest data suggesting a role for galectin-3 in adaptive immunity came from studies with mice deficient in UDP-N-acetylglucosaminyltransferase V (Mgat5).53 These mice showed increased TCR activation, susceptibility to autoimmune disease, and enhanced Th1 response, all attributed to the inefficient formation of multivalent lattices of galectin-3 and N-glycans in the TCR complex. Thus, surface galectin-3-glycoprotein lattices could play an inhibitory role by maintaining the baseline of immunity in the off state, besides limiting the development of an exacerbated immune response and autoimmune process. Accordingly, Stillman et al54 reported the purification of TCRß chain and several other receptors, such as CD29, CD43, CD45, and CD71, by affinity to immobilized galectin-3. Thus, galectin-3 interaction with TCRß may contribute to setting the threshold for TCR response to antigen.

In our experimental model, we demonstrated that the T. gondii-infected gal3C/C mice mounted an amplified Th1-type immune response, which could be due to the alteration in the activation threshold of gal3C/C cells. However, unlike the Mgat5 model, we did not detect significant differences between the gal3C/C and gal3+/+ mice regarding T. gondii antigen-induced T-cell proliferation rates (E. S. Bernardes, L. P. Ruas, M. C. Roque-Barreira, unpublished results). On the other hand, up-regulation of IL-12 production seen in gal3C/C DCs argues for a special role for galectin-3 on DC function and could explain, at least in part, the enhanced Th1 response found in gal3C/C mice. Even though cells from gal3C/C mice also produced higher levels of IL-12p70, our results do not exclude the possibility that galectin-3 regulates the production of IL-23??another heterodimeric cytokine closely related in structure to IL-12 and composed of an identical p40 subunit of IL-12 and a second smaller subunit, p19.44 However, more studies should be undertaken to better evaluate the mechanisms involved and at which stage of the cell development or activation galectin-3 is a key regulator. Our results are coherent with those of a very recent report using a murine model of asthma,24 showing that gal3C/C mice develop a lower Th2 response but a higher Th1 response when compared with gal3+/+ mice, leading the authors to suggest that galectin-3 regulates both Th1 and Th2 response. In addition, they found higher levels of antigen-specific IgG2a in gal3C/C mice, corroborating the hypothesis of an enhanced Th1 response.

We show here, for the first time, that galectin-3 suppresses the production of IL-12 by dendritic cells. Because its absence was associated with the production of higher levels of Th1 cytokines when these cells are stimulated, we suggest that galectin-3 may act in the regulatory loops controlling Th1 cytokine production. As a consequence, the development of an exacerbated cell-mediated immune response would be prevented. This novel information should be considered in the design of therapeutic interventions in autoimmune, inflammatory, and parasitic diseases.

Acknowledgements

We thank Vani M.A. Correa, Izilda R. Violante, Sandra M.O. Thomaz, Patricia E. Vendruscolo, and Lisia M. Esper for excellent technical assistance and Julio A. Siqueira, Savio H.F. Miranda, and Ednelson A. Mazzotto for expert animal care. We also thank Dr. Julio Aliberti for helpful discussions.

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作者单位：From the Departamento de Biologia Celular e Molecular e Bioagentes Patog?nicos,* Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; the Departamento de Imunoparasitologia, Universidade Federal de Uberlândia-ICBIM, Uberlândia,