I-Chun Weng

Galectin-3 negatively regulates TCR-mediated CD4+ T-cell activation at the immunological synapse

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3−/−) CD4+ T cells secreted more IFN-γ and IL-4 than gal3+/+CD4+ T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3+/+ T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3−/− T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alixs function at the IS.

Galectins as bacterial sensors in the host innate response

A number of galectin family members have been shown to play important roles in host defense against pathogens, and they are expressed by barrier tissues as well as immune cells. Galectins are present in the cytoplasm, nucleus, as well as extracellular space, and can function both inside and outside the cells. Galectins have been shown to bind to the surfaces of some pathogens and products released by the pathogens. These can result in either direct effects on growth of the pathogens or immune responses against them. Galectins may also affect the process of bacteria entering the host cells, such as adhesion. While galectin-mediated sensing of bacterial infection demonstrated so far mainly takes place at the extracellular site, it can occur at the intracellular site, intracellular galectins can recognize some intracellular bacteria. In the latter case, galectins may bind to glycans on the surface of the bacteria or the host glycans displayed on the ruptured membranes of endosomes that initially contain the bacteria. Thus, galectins can play important roles inside the cells in response to infection by intracellular bacteria.

Using carbon nanotube probes for high-resolution three-dimensional imaging of cells

While atomic force microscopy (AFM) has become a promising tool for visualizing membrane morphology of cells, many studies have reported the presence of artifacts such as cliffs on the edges of cells. These artifacts shield important structural features such as lamellopodia, filopodia, microvilli and membrane ridges, which represent characteristic status in signaling processes such as spreading and activation. These cliff-like edges arise from a premature contact of the probe side contact with the cell prior to the probe top apex-cell contact. Carbon nanotube (CNT) modified AFM probes were utilized to address this drawback. Using rat basophilic leukemia (RBL) cells, this work revealed that CNT probes diminish cliff-like artifacts and enabled visualization of entire membrane morphology and structural features in three dimensions. The high aspect ratio of CNT probes provides a very effective remedy to the cliff-like artifacts as well as tip convolution of conventional probes, which shall enhance the validity and application of AFM in cellular biology research.

Engineered Nanostructures of Antigen Provide an Effective Means for Regulating Mast Cell Activation

Nanostructures containing 2,4-dinitrophenyl (DNP) as antigen were designed and produced to investigate antibody-mediated activation of mast cells. The design consists of nanogrids of DNP termini inlaid in alkanethiol self-assembled monolayers (SAMs). Using scanning probe-based nanografting, nanometer precision was attained for designed geometry, size, and periodicity. Rat basophilic leukemia (RBL) cells exhibited high sensitivity to the geometry and local environment of DNP presented on these nanostructures. The impact included cellular adherence, spreading, membrane morphology, cytoskeleton structure, and activation. The highest level of spreading and activation was induced by nanogrids of 17 nm line width and 40 nm periodicity, with DNP haptens 1.4 nm above the surroundings. The high efficacy is attributed to two main factors. First, DNP sites in the nanostructure are highly accessible by anti-DNP IgE during recognition. Second, the arrangement or geometry of DNP termini in nanostructures promotes clustering of FcεRI receptors that are prelinked to IgE. The clustering effectively initiates Lyn-mediated signaling cascades, ultimately leading to the degranulation of RBL cells. This work demonstrates an important concept: that nanostructures of ligands provide new and effective cues for directing cellular signaling processes.

Examination of Galectins in Phagocytosis

Galectins, a family of β-galactoside-binding proteins, are expressed in many different phagocytic leukocytes (granulocytes, monocytes, and macrophages). A number of family members have been shown to play an important role in ingestion of particles (phagocytosis), thus contributing to clearance of damaged cells and host defense against pathogens. Here we describe procedures for analysis of the roles of galectins in phagocytosis by using galectin-3 as an example. We emphasize the function of endogenous galectin-3 as determined by comparison of phagocytosis by macrophages from galectin-3 knockout mice and wild-type mice. We focus on the role of galectin-3 in phagocytosis of pathogens and Fcγ receptor-mediated phagocytosis of opsonized cells and particles.

Galectin-3 Enhances Avian H5N1 Influenza A Virus–Induced Pulmonary Inflammation by Promoting NLRP3 Inflammasome Activation

Highly pathogenic avian influenza A H5N1 virus causes pneumonia and acute respiratory distress syndrome in humans. Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a β-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here, we describe galectin-3 up-regulation in mouse lung tissue after challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable with wild-type (WT) mice after infections. Compared with infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced IL-1β levels in bronchoalveolar lavage fluid. In addition, the bone marrow-derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains and secreted less IL-1β compared with BMMs from WT mice. However, similar levels of the inflammasome component of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) were observed in two genotypes of BMMs. Co-immunoprecipitation data indicated galectin-3 and NLRP3 interaction in BMMs infected with H5N1. An association was also observed between galectin-3 and NLRP3/apoptosis-associated speck-like proteins containing caspase-associated recruitment domain complex. Combined, our results suggest that endogenous galectin-3 enhances the effects of H5N1 infection by promoting host inflammatory responses and regulating IL-1β production by macrophages via interaction with NLRP3.

Galectin-9 Is Critical for Mucosal Adaptive Immunity through the T Helper 17–IgA Axis

Impairment of the intestinal mucosal immunity significantly increases the risk of acute and chronic diseases. IgA plays a major role in humoral mucosal immunity to provide protection against pathogens and toxins in the gut. Here, we investigated the role of endogenous galectin-9, a tandem repeat-type β-galactoside-binding protein, in intestinal mucosal immunity. By mucosal immunization of Lgals9-/- and littermate control mice, it was found that lack of galectin-9 impaired mucosal antigen-specific IgA response in the gut. Moreover, Lgals9-/- mice were more susceptible to developing watery diarrhea and more prone to death in response to high-dose cholera toxin. The results indicate the importance of galectin-9 in modulating intestinal adaptive immunity. Furthermore, bone marrow chimera mice were established, and galectin-9 in hematopoietic cells was found to be critical for adaptive IgA response. In addition, immunized Lgals9-/- mice exhibited lower expression of Il17 and fewer T helper 17 (Th17) cells in the lamina propria, implying that the Th17-IgA axis is involved in this mechanism. Taken together, these findings suggest that galectin-9 plays a role in mucosal adaptive immunity through the Th17-IgA axis. By manipulating the expression or activity of galectin-9, intestinal mucosal immune response can be altered and may benefit the development of mucosal vaccination.