An adipose tissue galectin controls endothelial cell function via preferential recognition of 3-fucosylated glycans.

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.

Hedgehog signaling pathway mediates tongue tumorigenesis in wild-type mice but not in Gal3-deficient mice.

Oral squamous cell carcinoma (OSCC) is one of the most aggressive cancers of the oral cavity and an important cause of death worldwide. Currently, there are limited clinical tools aiding clinicians to establish its early diagnosis, and genetic and epigenetic events leading to the pathogenesis of OSCC remain unsolved. The use of carcinogen-induced knocked out mouse models would help to improve its early detection and also determine the role of proteins such as galectin-3 (Gal3) in this process. Here we used a mouse model of oral carcinogenesis employing two mouse genotypes: wild-type (Gal3+/+) and galectin-3-deficient mice (Gal3-/-) challenged by the carcinogen 4NQO for 16 weeks. After induction, the expression of Wnt1, Wnt3A, Shh and Gli3 proteins in tongue samples was evaluated using an immunohistochemistry approach. All samples of dysplasia and carcinoma were negative for Wnt1. Wnt3A expression was detected in both Gal3+/+ and Gal3-/- mice, at similar levels. Wnt3A expression did not predict tongue tumorigenesis in either genotype. Dysplastic- and carcinoma-expressing Shh was statistically significantly higher in Gal3+/+ mice than Gal3-/- mice (p<0.0001), and was associated with tongue tumorigenesis only in the former. Gli3 expression decreased and increased from dysplasia to carcinoma in Gal3+/+ and Gal3-/- mice, respectively, although the difference was not significant. The results suggest that activated Wnt signaling is present in both mice, and that the Hh signaling pathway might play a role in tongue carcinoma development in Gal3+/+ mice.

Expression of APC protein during tongue malignant transformation in galectin-3-deficient mice challenged by the carcinogen 4-nitroquniline-n-oxide.

Galectin-3 (Gal3) has been implicated in the development of different tumors because of its involvement in the Wnt signaling pathway by promoting beta-catenin translocation into the nucleus. The APC protein, a negative regulator of this pathway, has been strongly implicated in the development of colon cancer, but still has an undetermined role in the formation of oral cancer. Therefore, this study aimed to evaluate the relationship between Gal3, the Wnt signaling pathway, and APC expression in dysplasias and carcinomas developed experimentally in mice. Sixty galectin-3-deficient (Gal3(-/-)) and 60 wild-type (Gal3(+/+)) mice were early employed to be treated with the carcinogen 4NQO for 16 weeks and killed at either week 16 or week 32. Tongues were removed, processed and embedded in paraffin blocks. Sections 5 µm thick were made, and then stained by H&E to establish the diagnosis of dysplasia and carcinoma. Sections of 2 µm thickness were made to detect APC expression in these lesions by immunohistochemistry. Oral carcinogenesis occurred in both groups of mice, but no statistical difference was reached. APC expression was exclusively seen in the cytoplasm of all lesions studied. In the intragroup analysis, the majority of dysplasias and carcinomas exhibiting higher APC immunoreactivity was observed in Gal3(-/-) mice compared to Gal3(+/+) mice, but no significant difference was found. However, a statistical difference was only observed between dysplastic lesions from two mice. Our results showed that neither the absence of Gal3 nor the APC protein appears to play a role in malignant transformation of the tongue.

Galectin-3 negatively regulates the frequency and function of CD4(+) CD25(+) Foxp3(+) regulatory T cells and influences the course of Leishmania major infection.

Galectin-3, an endogenous glycan-binding protein, plays essential roles during microbial infection by modulating innate and adaptive immunity. However, the role of galectin-3 within the CD4(+) CD25(+) Foxp3(+) T regulatory (TREG ) cell compartment has not yet been explored. Here, we found, in a model of Leishmania major infection, that galectin-3 deficiency increases the frequency of peripheral TREG cells both in draining lymph nodes (LNs) and sites of infection. These observations correlated with an increased severity of the disease, as shown by increased footpad swelling and parasite burden. Galectin-3-deficient (Lgals3(-/-) ) TREG cells displayed higher CD103 expression, showed greater suppressive capacity, and synthesized higher amounts of IL-10 compared with their wild-type (WT) counterpart. Furthermore, both TREG cells and T effector (TEFF ) cells from Lgals3(-/-) mice showed higher expression of Notch1 and the Notch target gene Hes-1. Interestingly, Notch signaling components were also altered in both TREG and TEFF cells from uninfected Lgals3(-/-) mice. Thus, endogenous galectin-3 regulates the frequency and function of CD4(+) CD25(+) Foxp3(+) TREG cells and alters the course of L. major infection.

The inactive form of glycogen synthase kinase-3ß is associated with the development of carcinomas in galectin-3 wild-type mice, but not in galectin-3-deficient mice.

Galectin-3 has been implicated in the tumor development via its mediation of the Wnt signaling pathway. Likewise, glycogen synthase kinase-3beta (GSK3ß) also plays a role in the Wnt signaling pathway by controlling the levels of cytoplasmic beta-catenin. Altered GSK3ß expression has been described in various tumors, but to date, there are no studies evaluating its expression in models of oral carcinogenesis. Additionally, it is unknown whether the absence of galectin-3 regulates the expression of GSK3ß. To this end, Gal3-deficient (Gal3(-/-)) and wild-type (Gal3(+/+)) male mice were treated with 4NQO for 16 weeks and sacrificed at week 16 and 32. The tongues were removed, processed, and stained with H&E to detect dysplasias and carcinomas. An immunohistochemical assay was performed to determine the level of P-GSK3ß-Ser9 expression in both groups. Carcinomas were more prevalent in Gal3(+/+) than Gal3(-/-) mice (55.5% vs. 28.5%), but no statistical difference was reached. In the dysplasias, the proportion of cells positive for P-GSK3ß-Ser9 was slightly higher in Gal3(+/+) than Gal3(-/-) mice (63% vs. 61%). In the carcinomas, a significant difference between Gal3(+/+) and Gal3(-/-) mice was found (74% vs. 59%; p=0.02). P-GSK3ß-Ser9-positive cells slightly decreased from the progression of dysplasias to carcinomas in Gal3(-/-) mice (61% vs. 59%; p>0.05). However, a significant increase in P-GSK3ß-Ser9 expression was observed from dysplasias to carcinomas in Gal3(+/+) mice (63% vs. 74%; p=0.01). In conclusion, these findings suggest that fully malignant transformation of the tongue epithelium is associated with increased P-GSK3ß-Ser9 expression in Gal3(+/+) mice, but not in Gal3(-/-) mice.

The involvement of the spleen during chronic phase of Schistosoma mansoni infection in galectin-3-/- mice.

Schistosoma mansoni synthesizes glycoconjugates which interact with galectin-3, eliciting an intense humoral immune response. Moreover, it was demonstrated that galectin-3 regulates B cell differentiation into plasma cells. Splenomegaly is a hallmark event characterized by polyclonal B cell activation and enhancement of antibody production. Here, we investigated whether galectin-3 interferes with spleen organization and B cell compartment during chronic schistosomiasis, using wild type (WT) and galectin-3-/- mice. In chronically-infected galectin-3-/- mice the histological architecture of the spleen, including white and red pulps, was disturbed with heterogeneous lymphoid follicles, an increased number of plasma cells (CD19-B220-/lowCD138+) and a reduced number of macrophages (CD19-B220-Mac-1+CD138-) and B lymphocytes (CD19+B220+/highCD138-), compared with the WT infected mice. In the absence of galectin-3 there was an increase of annexin-V+PI- cells and a major presence of apoptotic cells in spleen compared with WT infected mice. In spleen of WT infected mice galectin-3 was largely expressed in lymphoid follicles and extrafollicular sites. Thus, we propose that galectin-3 plays a role in splenic architecture, controlling distinct events such as apoptosis, macrophage activity, B cell differentiation and plasmacytogenesis in the course of S. mansoni infection.

The Promigratory Activity of the Matricellular Protein Galectin-3 Depends on the Activation of PI-3 Kinase

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-32/2 mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-32/2 sarcoma cells were more adherent and less migratory than galectin-3+/+ sarcomacells on laminin-111. When galectin-3 was transiently expressed in galectin-32/2 sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAKand phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 wasinhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111.

Activation of the Wnt/beta-catenin signaling pathway during oral carcinogenesis process is not influenced by the absence of galectin-3 in mice.

BACKGROUND/AIM: Galectin-3 has been associated with activated Wnt pathway, translocating beta-catenin into the nucleus. However, it is still unknown whether this lectin drives the Wnt signaling activation in lesions from galectin-3-deficient (Gal3⁻/⁻) mice. The purpose was to study beta-catenin expression in tongue lesions from Gal3⁻/⁻ and wild-type (Gal3⁺/⁺) mice and the status of Wnt signaling. MATERIALS AND METHODS: Twenty Gal3⁻/⁻ and Gal3⁺/⁺ male mice were challenged with 4-nitroquinolin-1-oxide and killed at week 16 and 32. Tongues were processed and stained with H&E to detect dysplasias and carcinomas. An imunohistochemical assay was performed to evaluate beta-catenin expression. RESULTS: Carcinomas were more evident in Gal3⁺/⁺ than Gal3⁻/⁻ mice (55.5% vs. 28.5%, respectively; p>0.05). Elevated expression of non-membranous beta-catenin was observed in dysplasias and carcinomas from both groups (p>0.05). CONCLUSION: Absence of galectin-3 does not interfere in the pattern of beta-catenin expression and therefore in the mediation of the Wnt signaling pathway.

Lack of galectin-3 disturbs mesenteric lymph node homeostasis and B cell niches in the course of Schistosoma mansoni infection.

Galectin-3 is a ß-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAcß1-4(Fucα1-3)GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine ß1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in non-lymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S.mansoni.

Absence of galectin-3 does not affect the development of experimental tongue carcinomas in mice.

BACKGROUND: Galectin-3 is a lectin that presents pivotal roles in tumor biology and there are no studies evaluating their expression in dysplasias and carcinomas developed from tongue carcinogenesis models. AIMS: To investigate the role of galectin-3 in the development of tongue carcinomas using a mouse model of oral carcinogenesis. METHODS: Galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice were challenged with 4-nitroquinoline-1-oxide in drinking water for 16weeks and killed at different times. Tongues were removed and the number of dysplasias and carcinomas was counted. An immunohistochemical study for galectin-3 was performed only in the tongue from gal3(+/+) mice. RESULTS: In both groups, a reduction of dysplasias and an increase of carcinomas from week 16 to week 32 (p>0.05) were observed. A predominance of high cytoplasmic and nuclear galectin-3 expression was observed in carcinomas (64.7%) and dysplasias (55.5%), respectively (p>0.05). The perilesional areas always presented a statistical cytoplasmic and nuclear galectin-3 overexpression. CONCLUSIONS: Absence of galectin-3 did not directly affect the process of carcinogenesis and a cytoplasm shift of galectin-3 seems to be associated with development of tongue carcinomas.

The promigratory activity of the matricellular protein galectin-3 depends on the activation of PI-3 kinase.

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3(-/-) mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3(-/-) sarcoma cells were more adherent and less migratory than galectin-3(+/+) sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3(-/-) sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111.

Galectin-3 plays a modulatory role in the life span and activation of murine neutrophils during early Toxoplasma gondii infection.

Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (Nphi) from C57BL/6 wild-type (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MTT assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in Nphi culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in Nphi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Nphi number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates T. gondii-induced Nphi toxicity as well as Nphi degranulation regardless of infection. Furthermore, Gal-3 expression by Nphi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in Nphi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in Nphi life span and activation during early T. gondii infection.

Galectin-3 regulates peritoneal B1-cell differentiation into plasma cells.

Extracellular galectin-3 participates in the control of B2 lymphocyte migration and adhesion and of their differentiation into plasma cells. Here, we analyzed the role of galectin-3 in B1-cell physiology and the balance between B1a and B1b lymphocytes in the peritoneal cavity. In galectin-3(-/-) mice, the total number of B1a lymphocytes was lower, while B1b lymphocyte number was higher as compared to wild-type mice. The differentiation of B1a cells into plasma cells was associated with their abnormal adhesion and location on the mesentery. The B220 and CD43, constitutively expressed by B1 lymphocytes, were respectively up- and downregulated in galectin-3(-/-) mice. Mononuclear cells were strongly adhered to the mesenteric membranes of both CD43(-/-) and galectin-3(-/-) mice, but in contrast to CD43(-/-) mice, the accumulation of B1 cells in peritoneal membranes in galectin-3(-/-) mice was accompanied by their functional differentiation into plasma cells. We have shown that in the absence of galectin-3, B1-cell differentiation into plasma cells is favored and the dynamic equilibrium of B1-cell populations in the peritoneum is maintained through a compensatory increase in B1b lymphocytes.

Lack of galectin-3 drives response to Paracoccidioides brasiliensis toward a Th2-biased immunity.

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection.

Galectin-3 is a beta-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3-/-) and their wild-type counterpart (gal3+/+) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3+/+ mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3+/+ macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1beta production and thus affecting resistance to R. equi infection.

Altered expression of galectin-3 induces cortical thymocyte depletion and premature exit of immature thymocytes during Trypanosoma cruzi infection.

During acute infection with Trypanosoma cruzi, the causative agent of Chagas' disease, the thymus undergoes intense atrophy followed by a premature escape of CD4+CD8+ immature cortical thymocytes. Here we report a pivotal role for the endogenous lectin galectin-3 in accelerating death of thymocytes and migration of these cells away from the thymus after T. cruzi infection. We observed a pronounced increase in galectin-3 expression that paralleled the extensive depletion of CD4+CD8+ immature thymocytes after infection. In vitro, recombinant galectin-3 induced increased levels of death in cortical immature thymocytes. Consistent with the role of galectin-3 in promoting cell death, thymuses from gal-3-/- mice did not show cortical thymocyte depletion after parasite infection in vivo. In addition, galectin-3 accelerated laminin-driven CD4+CD8+ thymocyte migration in vitro and in vivo induced exportation of CD4+CD8+ cells from the thymus to the peripheral compartment. Our findings provide evidence of a novel role for galectin-3 in the regulation of thymus physiology and identify a potential mechanism based on protein-glycan interactions in thymic atrophy associated with acute T. cruzi infection.

Galectin-3 mediates IL-4-induced survival and differentiation of B cells: functional cross-talk and implications during Trypanosoma cruzi infection.

The role of transcription factors in B cell survival and differentiation has been delineated during the last years. However, little is known about the intermediate signals and the intracellular pathways that control these events. In this study, we provide evidence both in vitro and in vivo, showing that galectin-3 (Gal-3), a beta-galactoside-binding protein, is a critical mediator of B cell differentiation and survival. Although Gal-3 is not expressed in resting B cells from normal mice, its expression is markedly induced after activation with stimuli such as IL-4 and CD40 cross-linking. These signals promote survival and block the final differentiation of these cells, thus allowing the rising of a memory B cell phenotype. In addition, Gal-3 is expressed in B cells from Trypanosoma cruzi-infected mice, which received signals for activation and differentiation in vivo. By using an antisense strategy, we determined that Gal-3 is a critical signal mediating the effects of IL-4 on B cell fate. Blockade of intracellular Gal-3 in vitro abrogated IL-4-induced survival of activated B cells, favoring the differentiation toward a plasma cell pathway. Moreover, B cells with restrained endogenous Gal-3 expression failed to down-regulate the Blimp-1 transcription factor after IL-4 stimulation. Finally, inhibition of Gal-3 in vivo skewed the balance toward plasma cell differentiation, which resulted in increased Ig production and parasite clearance during T. cruzi infection. Thus, the present study provides evidence of a novel role for Gal-3 as an intracellular mediator of B cell survival and a checkpoint in IL-4-induced B cell commitment toward a memory phenotype.

Galectins and their ligands: amplifiers, silencers or tuners of the inflammatory response?

Recent evidence has implicated galectins and their ligands as master regulators of immune cell homeostasis. Whereas some members of this family, such as galectin-3, behave as amplifiers of the inflammatory cascade, others, such as galectin-1, trigger homeostatic signals to shut off T-cell effector functions. These carbohydrate-binding proteins, identified by shared consensus amino acid sequences and affinity for beta-galactoside-containing sugars, participate in the homeostasis of the inflammatory response, either by regulating cell survival and signaling, influencing cell growth and chemotaxis, interfering with cytokine secretion, mediating cell-cell and cell-matrix interactions or influencing tumor progression and metastasis. The current wealth of new information promises a future scenario in which individual members of the galectin family or their ligands will be used as powerful anti-inflammatory mediators and selective modulators of the immune response.