Podoplanin-Expressing Macrophages Promote Lymphangiogenesis and Lymphoinvasion in Breast Cancer.

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin ß1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin ß1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis.

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

BACKGROUND: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. METHODS/PRINCIPLE FINDINGS: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. CONCLUSION/SIGNIFICANCE: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.

Interleukin-6 signalling mediates Galectin-8 co-stimulatory activity of antigen-specific CD4 T-cell response.

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-6-deficient antigen-presenting cells. In addition, exogenous IL-6 fully restored Gal-8-induced co-stimulation. Taken together, our results demonstrate that IL-6 signalling mediates the Gal-8 immune-stimulatory effect.

Galectin-8 activates dendritic cells and stimulates antigen-specific immune response elicitation.

Galectin-8 (Gal-8) is a mammalian ß-galactoside-binding lectin, endowed with proinflammatory properties. Given its capacity to enhance antigen-specific immune responses in vivo, we investigated whether Gal-8 was also able to promote APC activation to sustain T cell activation after priming. Both endogenous [dendritic cells (DCs)] and bone marrow-derived DCs (BMDCs) treated with exogenous Gal-8 exhibited a mature phenotype characterized by increased MHC class II (MHCII), CD80, and CD86 surface expression. Moreover, Gal-8-treated BMDCs (Gal-8-BMDCs) stimulated antigen-specific T cells more efficiently than immature BMDCs (iBMDCs). Proinflammatory cytokines IL-3, IL-2, IL-6, TNF, MCP-1, and MCP-5, as well as growth factor G-CSF, were augmented in Gal-8-BMDC conditioned media, with IL-6 as the most prominent. Remarkably, BMDCs from Gal-8-deficient mice (Lgals8-/- BMDC) displayed reduced CD86 and IL-6 expression and an impaired ability to promote antigen-specific CD4 T cell activation. To test if Gal-8-induced activation correlates with the elicitation of an effective immune response, soluble Gal-8 was coadministrated with antigen during immunization of BALB/cJ mice in the experimental foot-and-mouth disease virus (FMDV) model. When a single dose of Gal-8 was added to the antigen formulation, an increased specific and neutralizing humoral response was developed, sufficient to enhance animal protection upon viral challenge. IL-6 and IFN-γ, as well as lymphoproliferative responses, were also incremented in Gal-8/antigen-immunized animals only at 48 h after immunization, suggesting that Gal-8 induces the elicitation of an inflammatory response at an early stage. Taking together, these findings argue in favor of the use of Gal-8 as an immune-stimulator molecule to enhance the adaptive immune response.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.

Control of angiogenesis by galectins involves the release of platelet-derived proangiogenic factors.

Platelets contribute to vessel formation through the release of angiogenesis-modulating factors stored in their α-granules. Galectins, a family of lectins that bind ß-galactoside residues, are up-regulated in inflammatory and cancerous tissues, trigger platelet activation and mediate vascularization processes. Here we aimed to elucidate whether the release of platelet-derived proangiogenic molecules could represent an alternative mechanism through which galectins promote neovascularization. We show that different members of the galectin family can selectively regulate the release of angiogenic molecules by human platelets. Whereas Galectin (Gal)-1, -3, and -8 triggered vascular endothelial growth factor (VEGF) release, only Gal-8 induced endostatin secretion. Release of VEGF induced by Gal-8 was partially prevented by COX-1, PKC, p38 and Src kinases inhibitors, whereas Gal-1-induced VEGF secretion was inhibited by PKC and ERK blockade, and Gal-3 triggered VEGF release selectively through a PKC-dependent pathway. Regarding endostatin, Gal-8 failed to stimulate its release in the presence of PKC, Src and ERK inhibitors, whereas aspirin or p38 inhibitor had no effect on endostatin release. Despite VEGF or endostatin secretion, platelet releasates generated by stimulation with each galectin stimulated angiogenic responses in vitro including endothelial cell proliferation and tubulogenesis. The platelet angiogenic activity was independent of VEGF and was attributed to the concerted action of other proangiogenic molecules distinctly released by each galectin. Thus, secretion of platelet-derived angiogenic molecules may represent an alternative mechanism by which galectins promote angiogenic responses and its selective blockade may lead to the development of therapeutic strategies for angiogenesis-related diseases.

Diagnostic peptide discovery: prioritization of pathogen diagnostic markers using multiple features.

The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete "peptidomes". Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked [Formula: see text]10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes.

Binding of galectin-1 to αIIbß3 integrin triggers "outside-in" signals, stimulates platelet activation, and controls primary hemostasis.

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin-1 (Gal-1), a ß-galactoside-binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal-1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti-α(IIb) subunit ofα(IIb)ß(3) integrin or platelets from patients with Glanzmann's thrombasthenia syndrome (α(IIb)ß(3) deficiency), identified this integrin as a functional Gal-1 receptor in platelets. Binding of Gal-1 to platelets triggered the phosphorylation of ß(3)-integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA(2)) release, and Ca(2+) mobilization. Not only soluble but also immobilized Gal-1 promoted platelet activation. Gal-1-deficient (Lgals1(-/-)) mice showed increased bleeding time (P<0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1(-/-) platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of α(IIb)ß(3) was also observed in Lgals1(-/-) platelets, indicating a failure in the "outside-in" signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin-integrin interactions, for regulating platelet activation.

Identification of galectins as novel regulators of platelet signaling and function.

Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis. However, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Platelets can be activated by soluble molecules including thrombin, TXA2 , adenosine diphosphate (ADP), and serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor and collagen. In this article, we review recent advances on the role of galectins in platelet physiology. By acting in either soluble or immobilized form, these glycan-binding proteins trigger platelet activation through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer.

A Trypanosoma cruzi antigen signals CD11b+ cells to secrete cytokines that promote polyclonal B cell proliferation and differentiation into antibody-secreting cells.

Microbial-induced polyclonal activation of B cells is a common event in several forms of infections, and is believed to play a crucial role both for enhancing the production of specific antibodies and for maintenance of B cell memory. Therefore, a major challenge in biomedical research is the identification of pathogen-derived products capable of rapidly mounting B cell expansion and differentiation. Here we report that glutamate dehydrogenase (GDH) stimulates polyclonal proliferation and differentiation of naive B cells. This stimulation was found to be T cell independent, but to absolutely require CD11b(+) cells. Moreover, we demonstrate that stimulation of CD11b(+) cells by GDH leads to the production of IL-6, IL-10 and B cell-activating factor (BAFF), all of which combine to powerfully induce B cell expansion. Importantly, IL-6 and IL-10 further drive B cell terminal differentiation into plasma cells by up-regulating critical transcription factors and immunoglobulin secretion. Our data provide the first evidence that a protozoan antigen can induce BAFF production by accessory cells, which in concert with other cytokines trigger polyclonal B cell activation.

A sexual dimorphism in intrathymic sialylation survey is revealed by the trans-sialidase from Trypanosoma cruzi.

Sialylation is emerging as an important issue in developing thymocytes and is considered among the most significant cell surface modifications, although its physiologic relevance is far from being completely understood. It is regulated by the concerted expression of sialyl transferases along thymocyte development. After in vivo administration of trans-sialidase, a virulence factor from the American trypanosomatid Trypanosoma cruzi that directly transfers the sialyl residue among macromolecules, we found that the alteration of the sialylation pattern induces thymocyte apoptosis inside the "nurse cell complex." This suggests a glycosylation survey in the development of the T cell compartment. In this study, we report that this thymocyte apoptosis mechanism requires the presence of androgens. No increment in apoptosis was recorded after trans-sialidase administration in females or in antiandrogen-treated, gonadectomized, or androgen receptor mutant male mice. The androgen receptor presence was required only in the thymic epithelial cells as determined by bone marrow chimeric mouse approaches. The presence of the CD43 surface mucin, a molecule with a still undefined function in thymocytes, was another absolute requirement. The trans-sialidase-induced apoptosis proceeds through the TNF-alpha receptor 1 deathly signaling leading to the activation of the caspase 3. Accordingly, the production of the cytokine was increased in thymocytes. The ability of males to delete thymocytes altered in their sialylation pattern reveals a sexual dimorphism in the glycosylation survey during the development of the T cell compartment that might be related to the known differences in the immune response among sexes.