No abstract

Galectin-3 is a member of the β-galactoside-binding animal lectin family expressed in various cell types, including mast cells. To determine the role of galectin-3 in the function of mast cells, we studied bone marrow-derived mast cells (BMMC) from wild-type (gal3+/+) and galectin-3-deficient (gal3−/−) mice. Cells from the two genotypes showed comparable expression of IgE receptor and c-Kit. However, upon activation by FcεRI cross-linkage, gal3−/− BMMC secreted a significantly lower amount of histamine as well as the cytokine IL-4, compared with gal3+/+ BMMC. In addition, we found significantly reduced passive cutaneous anaphylaxis reactions in gal3−/− mice compared with gal3+/+ mice. These results indicate that there is a defect in the response of mast cells in gal3−/− mice. Unexpectedly, we found that gal3−/− BMMC contained a dramatically lower basal level of JNK1 protein compared with gal3+/+ BMMC, which is probably responsible for the lower IL-4 production. The decreased JNK1 level in gal3−/− BMMC is accompanied by a lower JNK1 mRNA level, suggesting that galectin-3 regulates the transcription of the JNK gene or processing of its RNA. All together, these results point to an important role of galectin-3 in mast cell biology.

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.

Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin‐3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL‐17 signaling. In liver tissues from patients with PBC and dnTGF‐βRII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis‐associated speck like protein was induced, with the downs tream activation of caspase‐1 and IL‐1β. Inwild‐typehepaticmacrophages, deoxycholic acid induced the association of Gal3 and NLRP3with direct activation of the inflammasome, resulting in an increase in IL‐1β. Downstreamretinoid‐related orphan receptor CmRNA, IL‐17A, and IL‐17Fwere induced. In Gal3‐/‐ macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the pathogenesis of cholestatic liver injury, we generated dnTGF‐βRII/galectin‐3‐/ (dn/Gal3‐/) mice, which showed impaired inflammasome activation alongwith significantly improved inflammation and fibrosis. Taken together, our data point to a novel role of Gal3 as an initiator of inflammatory signaling in autoimmune cholangitis, mediating the activation of NLRP3 inflammasome and inducing IL‐17 proinflammatory cascades. These studies provide a rationale to target Gal3 in autoimmune cholangitis and potentially other cholestatic diseases.—Tian, J., Yang, G., Chen, H.‐Y., Hsu, D. K., Tomilov, A., Olson, K.A., Dehnad, A., Fish, S. R., Cortopassi, G.,Zhao, B., Liu, F.‐T., Gershwin, M.E., Török, N. J., Jiang, J. X. Galectin‐3 regulates inflammasome activation in cholestatic liver injury. FASEB J. 30, 4202–4213 (2016). http://www.fasebj.org

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 pre-linked 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.

No abstract

Galectins are β-galactoside-binding animal lectins primarily found in the cytosol, while their carbohydrate ligands are mainly distributed in the extracellular space. Cytosolic galectins are anticipated to accumulate on damaged endocytic vesicles through binding to glycans initially displayed on the cell surface and subsequently located in the lumen of the vesicles, and this can be followed by cellular responses. To facilitate elucidation of the mechanism underlying this process, we adopted a model system involving induction of endocytic vesicle damage with light that targets the endocytosed amphiphilic photosensitizer disulfonated aluminum phthalocyanine. We demonstrate that the levels of galectins around damaged endosomes are dependent on the composition of carbohydrates recognized by the proteins. By super resolution imaging, galectin-3 and galectin-8 aggregates were found to be distributed in distinct microcompartments. Importantly, galectin accumulation is significantly affected when cell surface glycans are altered. Furthermore, accumulated galectins can direct autophagy adaptor proteins toward damaged endocytic vesicles, which are also significantly affected following alteration of cell surface glycans. We conclude that cytosolic galectins control cellular responses reflect dynamic modifications of cell surface glycans.

Galectin-8, a beta-galactoside-binding lectin, is upregulated in the gastric tissues of rhesus macaques infected with Helicobacter pylori. In this study, we found that H. pylori infection triggers intracellular galectin-8 aggregation in human-derived AGS gastric epithelial cells, and that these aggregates colocalize with lysosomes. Notably, this aggregation is markedly reduced following the attenuation of host O-glycan processing. This indicates that H. pylori infection induces lysosomal damage, which in turn results in the accumulation of cytosolic galectin-8 around damaged lysosomes through the recognition of exposed vacuolar host O-glycans. H. pylori-induced galectin-8 aggregates also colocalize with autophagosomes, and galectin-8 ablation reduces the activation of autophagy by H. pylori. This suggests that galectin-8 aggregates may enhance autophagy activity in infected cells. We also observed that both autophagy and NDP52, an autophagy adapter, contribute to the augmentation of galectin-8 aggregation by H. pylori. Additionally, vacuolating cytotoxin A, a secreted H. pylori cytotoxin, may contribute to the increased galectin-8 aggregation and elevated autophagy response in infected cells. Collectively, these results suggest that H. pylori promotes intracellular galectin-8 aggregation, and that galectin-8 aggregation and autophagy may reciprocally regulate each other during infection.