Binding of Toxoplasma gondii Glycosylphosphatidylinositols to Galectin-3 Is Required for Their Recognition by Macrophages

Abstract

We showed that the production of tumor necrosis factor (TNF) α by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-like receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a β-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs shows that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of TNF-α induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages. We showed that the production of tumor necrosis factor (TNF) α by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-like receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a β-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs shows that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of TNF-α induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages. IntroductionGlycosylphosphatidylinositol (GPI) 4The abbreviations used are: GPIglycosylphosphatidylinositolCRDcarbohydrate recognition domainNDN-terminal domainRUresonance unitsSPRsurface plasmon resonanceTLRToll-like receptor. -anchored proteins dominate the surface of the Toxoplasma gondii tachyzoite (1Black M.W. Boothroyd J.C. Microbiol. Mol. Biol. Rev. 2000; 64: 607-623Crossref PubMed Scopus (360) Google Scholar, 2Lekutis C. Ferguson D.J. Grigg M.E. Camps M. Boothroyd J.C. Int. J. Parasitol. 2001; 31: 1285-1292Crossref PubMed Scopus (181) Google Scholar). We have shown that T. gondii GPIs, as well as their glycan and lipid moieties, induce the production of tumor necrosis factor (TNF)-α in macrophages through the activation of the transcription factor NF-κB (3Debierre-Grockiego F. Azzouz N. Schmidt J. Dubremetz J.F. Geyer H. Geyer R. Weingart R. Schmidt R.R. Schwarz R.T. J. Biol. Chem. 2003; 278: 32987-32993Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Toll-like receptors (TLRs) recognize microbial components leading to cytokine production and antimicrobial responses through NF-κB activation (4Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4379) Google Scholar). We have shown that both TLR2 and TLR4 are involved in the NF-κB-dependent signaling cascade leading to production of TNF-α by macrophages exposed to T. gondii GPIs (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). The membrane protein CD14 is a co-receptor that associates with TLR4 to form a receptor complex for bacterial lipopolysaccharide (LPS) (6Yang Z. Khemlani L.S. Dean D.F. Carter C.D. Slauson D.O. Bochsler P.N. J. Leukocyte Biol. 1994; 55: 483-488Crossref PubMed Scopus (27) Google Scholar). CD14 is involved in the recognition of lipoproteins from Mycobacterium tuberculosis by TLR2 (7Drage M.G. Pecora N.D. Hise A.G. Febbraio M. Silverstein R.L. Golenbock D.T. Boom W.H. Harding C.V. Cell. Immunol. 2009; 258: 29-37Crossref PubMed Scopus (120) Google Scholar). In addition, CD14 is necessary for the TLR2-dependent response of macrophages to GPIs of Trypanosoma cruzi (8Campos M.A. Almeida I.C. Takeuchi O. Akira S. Valente E.P. Procópio D.O. Travassos L.R. Smith J.A. Golenbock D.T. Gazzinelli R.T. J. Immunol. 2001; 167: 416-423Crossref PubMed Scopus (454) Google Scholar). In contrast, CD14 is not required for the signaling leading to production of TNF-α by macrophages in response to the GPIs of T. gondii (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). The association of galectin-3 with TLR2 was demonstrated on PMA-differentiated THP-1 human macrophages infected with Candida albicans (9Jouault T. El Abed-El Behi M. Martinez-Esparza M. Breuilh L. Trinel P.A. Chamaillard M. Trottein F. Poulain D. J. Immunol. 2006; 177: 4679-4687Crossref PubMed Scopus (190) Google Scholar), and immunofluorescence microscopy showed the co-expression of galectin-3 with TLR4 on bone marrow-derived macrophages (10Li Y. Komai-Koma M. Gilchrist D.S. Hsu D.K. Liu F.T. Springall T. Xu D. J. Immunol. 2008; 181: 2781-2789Crossref PubMed Scopus (116) Google Scholar). Galectin-3 is a lectin specific for β-galactosides, composed of a C-terminal carbohydrate-recognition domain (CRD) and an N-terminal domain (ND) (11Hsu D.K. Zuberi R.I. Liu F.T. J. Biol. Chem. 1992; 267: 14167-14174Abstract Full Text PDF PubMed Google Scholar).The galectin-3-dependent recruitment of effector cells may be an important mechanism of resistance to parasite infection. Indeed, high levels of galectin-3 were found in the granulomas surrounding eggs and worms during Schistosoma mansoni infection (12van den Berg T.K. Honing H. Franke N. van Remoortere A. Schiphorst W.E. Liu F.T. Deelder A.M. Cummings R.D. Hokke C.H. van Die I. J. Immunol. 2004; 173: 1902-1907Crossref PubMed Scopus (142) Google Scholar). In addition, galectin-3 co-localizes with GalNAcβ1–4GlcNAc expressed on the surface of eggs and can mediate GalNAcβ1–4GlcNAc recognition and phagocytosis by macrophages. This implicates GalNAcβ1–4GlcNAc as a molecular parasite pattern for galectin-3-mediated immune recognition. In galectin-3−/− mice infected with S. mansoni, the size of the granulomas surrounding eggs was significantly decreased compared with infected wild type mice (13Breuilh L. Vanhoutte F. Fontaine J. van Stijn C.M. Tillie-Leblond I. Capron M. Faveeuw C. Jouault T. van Die I. Gosset P. Trottein F. Infect. Immun. 2007; 75: 5148-5157Crossref PubMed Scopus (89) Google Scholar). Galectin-3 deficiency affects the number of splenic T and B cells in S. mansoni-infected mice, whereas it does not modulate the number of dendritic cells. T. gondii infection of wild type mice leads to an up-regulation of galectin-3 expression in various tissues (14Bernardes E.S. Silva N.M. Ruas L.P. Mineo J.R. Loyola A.M. Hsu D.K. Liu F.T. Chammas R. Roque-Barreira M.C. Am. J. Pathol. 2006; 168: 1910-1920Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). T. gondii-infected galectin-3−/− mice develop reduced inflammatory responses in all organs, except for the lungs, and exhibit a higher parasite burden in the lungs and the brain (14Bernardes E.S. Silva N.M. Ruas L.P. Mineo J.R. Loyola A.M. Hsu D.K. Liu F.T. Chammas R. Roque-Barreira M.C. Am. J. Pathol. 2006; 168: 1910-1920Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Comparable survival rates were observed in galectin-3−/− and galectin-3+/+ mice orally infected with T. gondii. In contrast, higher mortality was observed in galectin-3−/− mice after intraperitoneal infection with T. gondii associated with a deficient influx of neutrophils and macrophages into the peritoneal cavity. Furthermore, a recent study indicates that galectin-3 has an important modulator function by interfering in the life span and activation of neutrophils early after their infection with T. gondii (15Alves C.M. Silva D.A. Azzolini A.E. Marzocchi-Machado C.M. Carvalho J.V. Pajuaba A.C. Lucisano-Valim Y.M. Chammas R. Liu F.T. Roque-Barreira M.C. Mineo J.R. Immunobiology. 2010; 215: 475-485Crossref PubMed Scopus (29) Google Scholar). In the present work, we show that the GPIs of T. gondii are ligands of galectin-3 and that the production of TNF-α induced by GPIs in macrophages requires the expression of galectin-3.DISCUSSIONGalectin-3 has more extended carbohydrate-recognition sites than galectin-1. This structural difference allows galectin-3 to bind to a wider range of oligosaccharide structures, including structures containing mannose (21Seetharaman J. Kanigsberg A. Slaaby R. Leffler H. Barondes S.H. Rini J.M. J. Biol. Chem. 1998; 273: 13047-13052Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). We identified the GPIs of T. gondii as ligands of galectin-3. KD values of galectin-3 for diacylglycerols and entire GPIs at the μm level and the nm level, respectively, were in the range of KD values of galectin-3 for other ligands, for example 3.6–6.1 μm for (Galβ1–3)n lactose, and 1.4 μm for biantennary N-linked oligosaccharide (22Pelletier I. Hashidate T. Urashima T. Nishi N. Nakamura T. Futai M. Arata Y. Kasai K. Hirashima M. Hirabayashi J. Sato S. J. Biol. Chem. 2003; 278: 22223-22230Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 23Sato S. Ouellet N. Pelletier I. Simard M. Rancourt A. Bergeron M.G. J. Immunol. 2002; 168: 1813-1822Crossref PubMed Scopus (208) Google Scholar, 24Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar). Six different GPIs are synthesized by the parasite. GPIs III and VI present a GalNAcβ1–4 branched on the first mannose of the evolutionary conserved core glycan, whereas GPIs I, II, IV, and V have an additional glucose residue linked with an α1–4 bond to the GalNAc. This glucose residue does not influence the recognition of the GPIs by galectin-3, because no difference was observed in the amounts of GPIs interacting with galectin-3 in the SPR assay. Binding of GalNAcβ1–4-containing glycans is not common to all galectin family members. Indeed, galectin-3 but not galectin-1 binds GalNAcβ1-4GlcNAc-glycans of S. mansoni (12van den Berg T.K. Honing H. Franke N. van Remoortere A. Schiphorst W.E. Liu F.T. Deelder A.M. Cummings R.D. Hokke C.H. van Die I. J. Immunol. 2004; 173: 1902-1907Crossref PubMed Scopus (142) Google Scholar). Galectin-3 can accommodate the O-2 N-acetyl moiety of the GalNAc residue, whereas a bulky histidine (at position 52) in galectin-1 may prevent GalNAc binding. Binding of galectin-3 to GalNAcβ1–4GlcNAc was completely prevented in the presence of lactose, supporting involvement of the CRD in this interaction (12van den Berg T.K. Honing H. Franke N. van Remoortere A. Schiphorst W.E. Liu F.T. Deelder A.M. Cummings R.D. Hokke C.H. van Die I. J. Immunol. 2004; 173: 1902-1907Crossref PubMed Scopus (142) Google Scholar). These results are in agreement with our observation that the synthetic glycan GPIa does not bind to galectin-1 but binds to the CRD of galectin-3, as evidenced by the lactose inhibitory effect. Galectin-3 recognizes the polygalactose structure, (Galβ1–3)n, found on lipophosphoglycan of Leishmania major (25Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Galectin-1 was not shown to have significant affinity for L. major, suggesting that the polygalactose epitope binds to the galectin-3-unique extended binding pocket, which is not present in galectin-1 (25Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The polygalactose is not expressed by Leishmania donovani. Thus, galectin-3 can distinguish between the two related parasites. In contrast, GPIs are expressed on both virulent (RH) and nonvirulent (PTG) strains of T. gondii, and the slight differences we have found in their structure 5S. Niehus, T. K. Smith, N. Azzouz, M. A. Compos, J. F. Dubremetz, D. Sibley, R. T. Schwarz, R. T. Gazzinelli, and F. Debierre-Grockiego, unpublished observations. did not affect the interaction with galectin-3 (KD = 0.2 nm, not shown).Galectin-1 binds to Davanat (composed of β1–4-linked d-mannopyranosyl units and d-galactopyranosyl residues attached via α1–6 linkage) with an apparent equilibrium dissociation constant of 10 μm, compared with 260 μm for lactose (26Miller M.C. Klyosov A. Mayo K.H. Glycobiology. 2009; 19: 1034-1045Crossref PubMed Scopus (65) Google Scholar). The β-galactoside-binding domain remains accessible in the galectin-1·Davanat complex because lactose can still bind with no apparent loss in affinity. Galectin-1 acts as a receptor for a protein, the tissue plasminogen activator. The interaction of tissue plasminogen activator with galectin-1 is higher than with galectin-3 (KD 9.1 μm and 17 mm, respectively) (27Roda O. Ortiz-Zapater E. Martínez-Bosch N. Gutiérrez-Gallego R. Vila-Perelló M. Ampurdanés C. Gabius H.J. André S. Andreu D. Real F.X. Navarro P. Gastroenterology. 2009; 136 (e1–e5): 1379-1390Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This illustrates that galectins interact not only with β-galactosides. Here, we show that the diacylglycerols of T. gondii GPIs interact strongly with galectin-1, which lacks a ND. Because the CRD of both galectins has affinities for different structures, further investigations are needed to conclude whether the lipid moiety of GPIs interacts with the CRD or the ND of galectin-3. We have shown previously that the diacylglycerols cleaved from the T. gondii GPIs are able to stimulate macrophages to produce TNF-α (3Debierre-Grockiego F. Azzouz N. Schmidt J. Dubremetz J.F. Geyer H. Geyer R. Weingart R. Schmidt R.R. Schwarz R.T. J. Biol. Chem. 2003; 278: 32987-32993Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) via signaling of both TLR2 and TLR4, whereas only the TLR2 signaling seems to be triggered by GPIa or the whole GPIs (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). The three-dimensional conformation of the complexes galectin-3·diacylglycerol and galectin-3·GPI (or GPIa) might determine their recognition or not by TLR4. Free GPIs are present in the in their entire by the parasites. and R. T. Schwarz, unpublished observations. We that the lipid interaction with galectin-3 and the may a major in cytokine amounts of inflammatory in galectin-3−/− cells (10Li Y. Komai-Koma M. Gilchrist D.S. Hsu D.K. Liu F.T. Springall T. Xu D. J. Immunol. 2008; 181: 2781-2789Crossref PubMed Scopus (116) Google Scholar). S. and T. gondii-infected galectin-3−/− mice a higher immune response and a decreased (14Bernardes E.S. Silva N.M. Ruas L.P. Mineo J.R. Loyola A.M. Hsu D.K. Liu F.T. Chammas R. Roque-Barreira M.C. Am. J. Pathol. 2006; 168: 1910-1920Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Because was associated with the production of higher levels of cells are it that galectin-3 may in the cytokine was shown that galectin-3−/− macrophages produce lower amounts of TNF-α after with Candida compared with galectin-3+/+ macrophages (9Jouault T. El Abed-El Behi M. Martinez-Esparza M. Breuilh L. Trinel P.A. Chamaillard M. Trottein F. Poulain D. J. Immunol. 2006; 177: 4679-4687Crossref PubMed Scopus (190) Google Scholar). We showed in the present study that galectin-3−/− macrophages are to produce TNF-α in response to T. gondii other than macrophages might be for the higher production of inflammatory observed in study indicates that the T. gondii TNF-α production both TLR2 and TLR4 in macrophages (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). CD14 is the well associated for the recognition of the ligand of TLR4. the CD14 is not required for the production of TNF-α by macrophages in response to the GPIs of T. gondii (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). We that galectin-3 is a protein involved in the response to the GPIs of T. gondii. is that galectin-3 interact with present in TLRs S. Am. J. Pathol. 2004; PubMed Scopus (65) Google Scholar). has demonstrated that the surface expression of TLR2 is on galectin-3−/− macrophages E.S. Ruas L.P. Loyola A.M. C. M.C. Hsu D.K. Liu F.T. Chammas R. Roque-Barreira M.C. J. Immunol. 2008; PubMed Scopus Google Scholar). This up-regulation to an production of TNF-α in response to the GPIs because the expression of galectin-3 is results show that galectin-1 is not involved in the TNF-α production by macrophages in response to galectin-1 was found to NF-κB activation macrophages P. S. T. R. Cell. 2010; PubMed Scopus Google Scholar). We that galectin-1 residues linked to glycans on TLR4 and can no In contrast, the TNF-α production was higher in compared with wild type macrophages suggesting that galectin-1 may the TLR4 signaling in response to T. gondii that galectin-1 is involved in the recognition of parasites. galectin-1 expression is in and in by T. and the expression of galectin-1 is in tissue from with L. S. Immunol. 2001; PubMed Scopus Google Scholar). Because the of T. cruzi to cells is by an of galectin-1 on tissue parasite this infection Cell. Microbiol. 2008; PubMed Scopus Google Scholar). Galectin-1 acts as a receptor for the by interacting with parasite at the surface of human cells Cell. Microbiol. 2008; PubMed Scopus Google Scholar). galectin-1 may be involved in the of T. gondii to the supporting by this the IntroductionGlycosylphosphatidylinositol (GPI) 4The abbreviations used are: GPIglycosylphosphatidylinositolCRDcarbohydrate recognition domainNDN-terminal domainRUresonance unitsSPRsurface plasmon resonanceTLRToll-like receptor. -anchored proteins dominate the surface of the Toxoplasma gondii tachyzoite (1Black M.W. Boothroyd J.C. Microbiol. Mol. Biol. Rev. 2000; 64: 607-623Crossref PubMed Scopus (360) Google Scholar, 2Lekutis C. Ferguson D.J. Grigg M.E. Camps M. Boothroyd J.C. Int. J. Parasitol. 2001; 31: 1285-1292Crossref PubMed Scopus (181) Google Scholar). We have shown that T. gondii GPIs, as well as their glycan and lipid moieties, induce the production of tumor necrosis factor (TNF)-α in macrophages through the activation of the transcription factor NF-κB (3Debierre-Grockiego F. Azzouz N. Schmidt J. Dubremetz J.F. Geyer H. Geyer R. Weingart R. Schmidt R.R. Schwarz R.T. J. Biol. Chem. 2003; 278: 32987-32993Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Toll-like receptors (TLRs) recognize microbial components leading to cytokine production and antimicrobial responses through NF-κB activation (4Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4379) Google Scholar). We have shown that both TLR2 and TLR4 are involved in the NF-κB-dependent signaling cascade leading to production of TNF-α by macrophages exposed to T. gondii GPIs (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). The membrane protein CD14 is a co-receptor that associates with TLR4 to form a receptor complex for bacterial lipopolysaccharide (LPS) (6Yang Z. Khemlani L.S. Dean D.F. Carter C.D. Slauson D.O. Bochsler P.N. J. Leukocyte Biol. 1994; 55: 483-488Crossref PubMed Scopus (27) Google Scholar). CD14 is involved in the recognition of lipoproteins from Mycobacterium tuberculosis by TLR2 (7Drage M.G. Pecora N.D. Hise A.G. Febbraio M. Silverstein R.L. Golenbock D.T. Boom W.H. Harding C.V. Cell. Immunol. 2009; 258: 29-37Crossref PubMed Scopus (120) Google Scholar). In addition, CD14 is necessary for the TLR2-dependent response of macrophages to GPIs of Trypanosoma cruzi (8Campos M.A. Almeida I.C. Takeuchi O. Akira S. Valente E.P. Procópio D.O. Travassos L.R. Smith J.A. Golenbock D.T. Gazzinelli R.T. J. Immunol. 2001; 167: 416-423Crossref PubMed Scopus (454) Google Scholar). In contrast, CD14 is not required for the signaling leading to production of TNF-α by macrophages in response to the GPIs of T. gondii (5Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. J. Immunol. 2007; 179: 1129-1137Crossref PubMed Scopus (216) Google Scholar). The association of galectin-3 with TLR2 was demonstrated on PMA-differentiated THP-1 human macrophages infected with Candida albicans (9Jouault T. El Abed-El Behi M. Martinez-Esparza M. Breuilh L. Trinel P.A. Chamaillard M. Trottein F. Poulain D. J. Immunol. 2006; 177: 4679-4687Crossref PubMed Scopus (190) Google Scholar), and immunofluorescence microscopy showed the co-expression of galectin-3 with TLR4 on bone marrow-derived macrophages (10Li Y. Komai-Koma M. Gilchrist D.S. Hsu D.K. Liu F.T. Springall T. Xu D. J. Immunol. 2008; 181: 2781-2789Crossref PubMed Scopus (116) Google Scholar). Galectin-3 is a lectin specific for β-galactosides, composed of a C-terminal carbohydrate-recognition domain (CRD) and an N-terminal domain (ND) (11Hsu D.K. Zuberi R.I. Liu F.T. J. Biol. Chem. 1992; 267: 14167-14174Abstract Full Text PDF PubMed Google Scholar).The galectin-3-dependent recruitment of effector cells may be an important mechanism of resistance to parasite infection. Indeed, high levels of galectin-3 were found in the granulomas surrounding eggs and worms during Schistosoma mansoni infection (12van den Berg T.K. Honing H. Franke N. van Remoortere A. Schiphorst W.E. Liu F.T. Deelder A.M. Cummings R.D. Hokke C.H. van Die I. J. Immunol. 2004; 173: 1902-1907Crossref PubMed Scopus (142) Google Scholar). In addition, galectin-3 co-localizes with GalNAcβ1–4GlcNAc expressed on the surface of eggs and can mediate GalNAcβ1–4GlcNAc recognition and phagocytosis by macrophages. This implicates GalNAcβ1–4GlcNAc as a molecular parasite pattern for galectin-3-mediated immune recognition. In galectin-3−/− mice infected with S. mansoni, the size of the granulomas surrounding eggs was significantly decreased compared with infected wild type mice (13Breuilh L. Vanhoutte F. Fontaine J. van Stijn C.M. Tillie-Leblond I. Capron M. Faveeuw C. Jouault T. van Die I. Gosset P. Trottein F. Infect. Immun. 2007; 75: 5148-5157Crossref PubMed Scopus (89) Google Scholar). Galectin-3 deficiency affects the number of splenic T and B cells in S. mansoni-infected mice, whereas it does not modulate the number of dendritic cells. T. gondii infection of wild type mice leads to an up-regulation of galectin-3 expression in various tissues (14Bernardes E.S. Silva N.M. Ruas L.P. Mineo J.R. Loyola A.M. Hsu D.K. Liu F.T. Chammas R. Roque-Barreira M.C. Am. J. Pathol. 2006; 168: 1910-1920Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). T. gondii-infected galectin-3−/− mice develop reduced inflammatory responses in all organs, except for the lungs, and exhibit a higher parasite burden in the lungs and the brain (14Bernardes E.S. Silva N.M. Ruas L.P. Mineo J.R. Loyola A.M. Hsu D.K. Liu F.T. Chammas R. Roque-Barreira M.C. Am. J. Pathol. 2006; 168: 1910-1920Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Comparable survival rates were observed in galectin-3−/− and galectin-3+/+ mice orally infected with T. gondii. In contrast, higher mortality was observed in galectin-3−/− mice after intraperitoneal infection with T. gondii associated with a deficient influx of neutrophils and macrophages into the peritoneal cavity. Furthermore, a recent study indicates that galectin-3 has an important modulator function by interfering in the life span and activation of neutrophils early after their infection with T. gondii (15Alves C.M. Silva D.A. Azzolini A.E. Marzocchi-Machado C.M. Carvalho J.V. Pajuaba A.C. Lucisano-Valim Y.M. Chammas R. Liu F.T. Roque-Barreira M.C. Mineo J.R. Immunobiology. 2010; 215: 475-485Crossref PubMed Scopus (29) Google Scholar). In the present work, we show that the GPIs of T. gondii are ligands of galectin-3 and that the production of TNF-α induced by GPIs in macrophages requires the expression of galectin-3.