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Toxoplasma gondii Infection Reveals a Novel Regulatory Role for Galectin-3 in the Interface of Innate and Adaptive Immunity
2006; Elsevier BV; Volume: 168; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.050636
ISSN1525-2191
AutoresEmerson Soares Bernardes, Neide Maria Silva, Luciana Pereira Ruas, José Roberto Mineo, A.M. Loyola, Daniel K. Hsu, Fu‐Tong Liu, Roger Chammas, Maria Cristina Roque‐Barreira,
Tópico(s)Parasitic Infections and Diagnostics
ResumoIn attempts to investigate the role of galectin-3 in innate immunity, we studied galectin-3-deficient (gal3−/−) 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 gal3−/− mice developed reduced inflammatory response in all of these organs but the lungs. Brain of gal3−/− mice displayed a significantly reduced number of infiltrating monocytes/macrophages and CD8+ cells and a higher parasite burden. Furthermore, gal3−/− 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 gal3−/− mice produced higher amounts of interleukin-12 than cells from gal3+/+ mice, possibly explaining the higher Th1 response verified in the gal3−/− 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. In attempts to investigate the role of galectin-3 in innate immunity, we studied galectin-3-deficient (gal3−/−) 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 gal3−/− mice developed reduced inflammatory response in all of these organs but the lungs. Brain of gal3−/− mice displayed a significantly reduced number of infiltrating monocytes/macrophages and CD8+ cells and a higher parasite burden. Furthermore, gal3−/− 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 gal3−/− mice produced higher amounts of interleukin-12 than cells from gal3+/+ mice, possibly explaining the higher Th1 response verified in the gal3−/− 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. Galectins are a family of animal lectins composed of 15 members that are conserved throughout animal evolution.1Barondes SH Cooper DNW Gitt MA Leffler H Galectins: structure and function of a large family of animal lectins.J Biol Chem. 1994; 269: 20807-20810Abstract Full Text PDF PubMed Google Scholar, 2Kasai K Hirabayashi J Galectins: a family of animal lectins that decipher glycocodes.J Biochem. 1996; 119: 1-8Crossref PubMed Scopus (458) Google Scholar, 3Hughes RC The galectin family of mammalian carbohydrate-binding molecules.Biochem Soc Trans. 1997; 25: 1194-1198Crossref PubMed Scopus (108) Google Scholar, 4Leffler H Carlsson S Hedlund M Qian Y Poirier F Introduction to galectins.Glycoconj J. 2004; 19: 433-440Crossref PubMed Scopus (535) Google Scholar 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.5Liu FT Regulatory roles of galectins in the immune response.Int Arch Allergy Immunol. 2005; 136: 385-400Crossref PubMed Scopus (166) Google Scholar They can also modulate cellular activities by functioning intracellularly.6Hsu DK Liu FT Regulation of cellular homeostasis by galectins.Glycoconj J. 2004; 19: 507-515Crossref PubMed Scopus (129) Google Scholar 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.7Liu FT Galectins: a new family of regulators of inflammation.Clin Immunol. 2000; 97: 79-88Crossref PubMed Scopus (191) Google Scholar Thus, in contrast to the anti-inflammatory effect of galectin-1, a powerful pro-inflammatory activity has been proposed for galectin-3. 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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-inflammatory22Colnot C Ripoche MA Milon G Montagutelli X Crocker PR Poirier F Maintenance of granulocyte numbers during acute peritonitis is defective in galectin-3-null mutant mice.Immunology. 1998; 94: 290-296Crossref PubMed Scopus (155) Google Scholar, 23Hsu DK Yang R-Y Yu L Pan Z Salomon DR Fung-Leung WP Liu FT Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses.Am J Pathol. 2000; 156: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 24Zuberi RI Hsu DK Kalayci O Chen HY Sheldon HK Yu L Apgar JR Kawakami T Lilly CM Liu FT Critical role for galectin-3 in airway inflammation and bronchial hyperresponsiveness in a murine model of asthma.Am J Pathol. 2004; 165: 2045-2053Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar and phagocytotic25Sano H Hsu DK Apgar JR Yu L Sharma BB Kuwabara I Izui S Liu FT Critical role of galectin-3 in phagocytosis by macrophages.J Clin Invest. 2003; 112: 389-397Crossref PubMed Scopus (344) Google Scholar roles of galectin-3 and for its function as a receptor of “advanced glycation end” products.26Iacobini C Menini S Oddi G Ricci C Amadio L Pricci F Olivieri A Sorcini M Di Mario U Pesce C Pugliese G Galectin-3/AGE-receptor 3 knockout mice show accelerated AGE-induced glomerular injury: evidence for a protective role of galectin-3 as an AGE receptor.FASEB J. 2004; 18: 1773-1775Crossref PubMed Scopus (97) Google Scholar 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 gal3−/− 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.27Yap GS Sher A Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function.Immunobiology. 1999; 201: 240-247Crossref PubMed Scopus (184) Google Scholar 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)-γ production driven by interleukin (IL)-12 derived from dendritic cells.27Yap GS Sher A Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function.Immunobiology. 1999; 201: 240-247Crossref PubMed Scopus (184) Google Scholar, 28Gazzinelli RT Hieny S Wynn TA Wolf S Sher A Interleukin 12 is required for the T-lymphocyte-independent induction of interferon gamma by an intracellular parasite and induces resistance in T-cell-deficient hosts.Proc Natl Acad Sci USA. 1993; 90: 6115-6119Crossref PubMed Scopus (761) Google Scholar, 29Reis e Sousa C Hieny S Scharton-Kersten T Jankovic D Charest H Germain RN Sher A In vivo microbial stimulation induces rapid CD40 ligand: independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas.J Exp Med. 1997; 186: 1819-1829Crossref PubMed Scopus (771) Google Scholar 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. The galectin-3-deficient (gal3−/−) mice were generated as previously described23Hsu DK Yang R-Y Yu L Pan Z Salomon DR Fung-Leung WP Liu FT Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses.Am J Pathol. 2000; 156: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar 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. The low-virulent ME-49 strain of T. gondii was used to infect the mice.30Lunde MN Jacobs L Antigenic differences between endozoites and cystozoites of Toxoplasma gondii.J Parasitol. 1983; 69: 806-808Crossref PubMed Scopus (92) Google Scholar 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.31Gazzinelli RT Hakin S Hieny S Shearer G Sher A Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-γ production and protective immunity induced by an attenuated Toxoplasma gondii vaccine.J Immunol. 1991; 146: 286-292Crossref PubMed Google Scholar The Ethics Committee on Animal Research of the University of Sao Paulo approved all procedures performed in the studies described herein. Gal3+/+ and gal3−/− 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 gal3−/− 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 × 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. 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)32Ho MK Springer TA Mac-2, a novel 32,000 Mr mouse macrophage subpopulation-specific antigen defined by monoclonal antibodies.J Immunol. 1982; 128: 1221-1228Crossref PubMed Google Scholar 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.33dos Santos PV Roffe E Santiago HC Torres RA Marino AP Paiva CN Silva AA Gazzinelli RT Lannes-Vieira J Prevalence of CD8(+) alpha beta T cells in Trypanosoma cruzi-elicited myocarditis is associated with acquisition of CD62 L(Low) LFA-1 (High) VLA-4 (High) activation phenotype and expression of IFN-gamma-inducible adhesion and chemoattractant molecules.Microbes Infect. 2001; 3: 971-984Crossref PubMed Scopus (91) Google Scholar Immunoperoxidase staining with polyclonal rabbit antibodies against T. gondii was also used for detection of the parasites.34Suzuki Y Yang Q Conley FK Abrams JS Remington JS Antibody against interleukin-6 reduces inflammation and numbers of cysts in brains of mice with toxoplasmic encephalitis.Infect Immun. 1994; 62: 2773-2778Crossref PubMed Scopus (57) Google Scholar The tissue parasitism detected by immunohistochemistry was scored by counting the total number of cyst-like structures and parasitophorous vacuoles in 40 microscopic fields (×400) per histopathological section (40-μm distance between sections) as described previously.35Silva NM Rodrigues CV Santoro MM Reis LFL Alvarez-Leite JI Gazzinelli RT Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon and requirement of interferon regulatory factor 1.Infect Immun. 2002; 70: 859-868Crossref PubMed Scopus (151) Google Scholar For the inflammatory infiltrate score, the total numbers of focal or diffuse inflammatory foci were counted in 25 microscope fields (×200) 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.35Silva NM Rodrigues CV Santoro MM Reis LFL Alvarez-Leite JI Gazzinelli RT Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon and requirement of interferon regulatory factor 1.Infect Immun. 2002; 70: 859-868Crossref PubMed Scopus (151) Google Scholar, 36Michailowsky V Silva NM Rocha CD Vieira LQ Lannes-Vieira J Gazzinelli RT Pivotal role of interleukin-12 and interferon-gamma axis in controlling tissue parasitism and inflammation in the heart and central nervous system during Trypanosoma cruzi infection.Am J Pathol. 2001; 159: 1723-1733Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar Four sections were counted for each animal, and individual data points were determined from the mean result of the four sections. The ME-49 T. gondii-infected gal3+/+ and gal3−/− 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 × 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-γ (10 ng/ml; BD PharMingen, San Diego, CA), or LPS + IFN-γ. The culture supernatants were collected after 48 hours for determination of cytokine levels, as described below. Dendritic cells (DCs) were purified from the spleen as previously described,37Crowley M Inaba K Witmer-Pack M Steinman RM The cell surface of mouse DC: FACS analyses of DC from different tissues including thymus.Cell Immunol. 1989; 118: 108-125Crossref PubMed Scopus (288) Google Scholar 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.38Uzonna JE Kaushik RS Zhang Y Gordon JR Tabel H Experimental murine Trypanosoma congolense infections: II. Role of splenic adherent CD3+Thy12+ TCR alpha beta- gamma delta- CD4+8− and CD3+Thy12+ TCR−alpha beta- gamma delta CD4−8− cells in the production of IL-4, IL-10, and IFN-gamma and in trypanosome elicited immunosuppression.J Immunol. 1998; 161: 6189-6197PubMed Google Scholar Splenic dendritic cells and macrophages from uninfected mice were both isolated as described above. Splenic DCs (2 × 105/well) and adherent macrophages (2 × 105/well) were cultured and stimulated for 48 hours with 5 μg/ml LPS (Sigma), 10 ng/ml IFN-γ (PharMingen), or LPS (5 μg/ml) plus IFN-γ (10 ng/ml). After incubation, the culture supernatants were collected, separated from cells by centrifugation, and stored at −70°C until analysis. The resulting IL-12p40 production was measured by enzyme-linked immunosorbent assay (ELISA), as described below. IL-12p40, IL-12p70, IFN-γ, 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-γ and IL-10. 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,39Turunen HJ Vuorio KA Leinikki PO Determination of IgG, IgM and IgA antibody responses in human toxoplasmosis by enzyme-linked immunosorbent assay (ELISA).Scand J Infect Dis. 1983; 15: 307-311Crossref PubMed Scopus (42) Google Scholar as follows: EI = (ODsample/ODcutt-off), where values of EI ≥1.0 were considered as cut-off titers. 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. 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). 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 gal3−/− mice were stained by the same anti-galectin-3 antibody (data not shown). Because oral infection by T. gondii is typically followed by proliferation of parasites in the small intestine40Liesenfeld O Oral infection of C57BL/6 mice with Toxoplasma gondii: a new model of inflammatory bowel disease?.J Infect Dis. 2002; 185: 96-101Crossref PubMed Scopus (119) Google Scholar and because susceptibility of C57BL/6 mice to oral infection is partially attributed to necrosis of the small intestinal villi,41Liesenfeld O Kosek J Remington JS Suzuki Y Association of CD4+ T cell-dependent, inferferon-gamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii.J Exp Med. 1996; 184: 597-607Crossref PubMed Scopus (280) Google Scholar we compared tissue parasitism and inflammatory response in the small intestine of the infected mice. In both gal3+/+ and gal3−/− 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 gal3−/− 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 gal3−/− 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 gal3−/− 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+/+
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