Ulf J. Nilsson

Regulation of Alternative Macrophage Activation by Galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-γ/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-β-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.

Regulation of Transforming Growth Factor-beta 1-driven Lung Fibrosis by Galectin-3

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. OBJECTIVES To examine the role of galectin-3 in pulmonary fibrosis. METHODS We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1-induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β-induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF. CONCLUSIONS This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.

Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Tubing loops as a model for cardiopulmonary bypass circuits: both the biomaterial and the blood-gas phase interfaces induce complement activation in an in vitro model.

We describe here a model for the study of blood/surface and blood/air interaction as encountered in cardiopulmonary bypass (CPB) circuits. Polyethylene tubing was filled with serum or blood and closed end to end into loops whereby the volume of the remaining air bubble was inversely varied with respect to that of the fluid. The loops were rotated vertically in a water bath at 37°C. The profiles of C3a, iC3, and TCC generation were similar to those observed at surgery, involving CPB. Soluble heparin and heparan sulfate inhibited both C3a and TCC formation, but surface-conjugated heparin had only a minor effect. Binding of C3 and/or C3 fragments to the heparin surface was much reduced compared to the amine matrix to which heparin was linked, but compared with the polyethylene surface the effect was less pronounced. These data suggest that, in addition to the biomaterial surface, the blood-gas interface seems to play an important role in the activation of complement and that this activation is inhibitable by high concentrations of soluble glucose aminoglycans.

Galectin inhibitory disaccharides promote tumour immunity in a breast cancer model

High level galectin-1 expression results in cancer cell evasion of the immune response, increased tumour survival and aggressive metastases. Using a galectin-1 polyclonal antibody, high levels of galectin-1 protein were shown to be expressed by breast cancer cells established from FVB/N MMTV-c-neu mice as well as by the B16F10 melanoma cell line. In mixed lymphocyte cultures using tumour cells as antigenic stimulators, addition of recombinant galectin-1 dose-dependently inhibited lymphocyte production. Disaccharides were identified that inhibited galectin-1 function and increased growth and activation of CD8(+) CTLs killing cancer cells. X-ray crystallographic structures of human galectin-1 in complex with inhibitory disaccharides revealed their mode of binding. Combining galectin-blocking carbohydrates as adjuvants with vaccine immunotherapy in vivo to promote immune responses significantly decreased tumour progression and improved the outcomes for tumour challenged mice. This is the first report showing that suitably selected galectin-1 blocking disaccharides will act as adjuvants promoting vaccine stimulated immune responses against tumours in vivo.

Short-chain fatty acid formation in the hindgut of rats fed oligosaccharides varying in monomeric composition, degree of polymerisation and solubility.

The contents of short-chain fatty acids were investigated in rats fed lactitol, lactulose and four fructo-oligosaccharides of different degree of polymerisation and solubility. Fructo-oligosaccharides with a low degree of polymerisation (2-8) generated the highest levels of butyric acid all along the hindgut, whereas fructo-oligosaccharides with a high degree of polymerisation (10-60) generated the highest levels of propionic acid. These specific differences were also generally reflected in the caecal pools and molar proportions of short-chain fatty acids. The lower solubility of the fructo-oligosaccharides was related to a lower degree of caecal fermentation. Lactulose and lactitol yielded high proportions of acetic acid and low proportions of butyric acid. It is concluded that both the degree of polymerisation and the solubility may affect short-chain fatty acid formation, whereas the fructose content per se seem to be of less importance. This may be of interest when designing foods with specific health effects.

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate–protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.

Ligand induced galectin-3 self-association

Background: One galectin-3 function is to bind glycoproteins and cross-link them. Results: A glycoprotein engaged many more galectin-3 carbohydrate-binding sites than its number of relevant glycans. Conclusion: The ligand induced binding of one galectin-3 to another galectin-3 to form oligomers in a previously unrecognized way. Significance: This differs from previous models and provides a new framework to interpret biological effects of galectin-3. Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (∼50 μm) as compared with wild type (∼1 μm). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-d-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>∼50 μm) in agreement with published results and perhaps due to type-N self-association.

Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human‐specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Galα4Gal‐containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Galα4Gal moiety, but parts of the GalNAcβ and glucose residues, which flank the Galα4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Galα4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human‐specific PapG in binding the GbO4 that dominates In the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG‐GbO4 interaction will make it possible to design antiadherence therapeutics.

Galectin‐3 deficiency prevents concanavalin A–induced hepatitis in mice

We used concanavalin A (Con A)‐induced liver injury to study the role of galectin‐3 (Gal‐3) in the induction of inflammatory pathology and hepatocellular damage. We tested susceptibility to Con A–induced hepatitis in galectin‐3‐deficient (Gal‐3−/−) mice and analyzed the effects of pretreatment with a selective inhibitor of Gal‐3 (TD139) in wild‐type (WT) C57BL/6 mice, as evaluated by a liver enzyme test, quantitative histology, mononuclear cell (MNC) infiltration, cytokine production, intracellular staining of immune cells, and percentage of apoptotic MNCs in the liver. Gal‐3−/− mice were less sensitive to Con A–induced hepatitis and had a significantly lower number of activated lymphoid and dendritic cells (DCs) in the liver. The level of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin (IL)‐17 and ‐4 in the sera and the number of TNFα‐, IFNγ‐, and IL‐17‐ and ‐4‐producing cluster of differentiation (CD)4+ cells as well as IL‐12‐producing CD11c+ DCs were lower, whereas the number of IL‐10‐producing CD4+ T cells and F4/80+ macrophages were significantly higher in livers of Gal‐3−/− mice. Significantly higher percentages of late apoptotic Annexin V+ propidium‐idodide+ liver‐infiltrating MNCs and splenocytes were observed in Gal‐3−/− mice, compared to WT mice. Pretreatment of WT C57BL/6 mice with TD139 led to the attenuation of liver injury and milder infiltration of IFNγ‐ and IL‐17‐ and ‐4‐producing CD4+ T cells, as well as an increase in the total number of IL‐10‐producing CD4+ T cells and F4/80+ CD206+ alternatively activated macrophages and prevented the apoptosis of liver‐infiltrating MNCs. Conclusions: Gal‐3 plays an important proinflammatory role in Con A–induced hepatitis by promoting the activation of T lymphocytes and natural killer T cells, maturation of DCs, secretion of proinflammatory cytokines, down‐regulation of M2 macrophage polarization, and apoptosis of MNCs in the liver. (HEPATOLOGY 2012;55:1954–1964)