Galectin-9 mediates neutrophil capture and adhesion in a CD44 and ß2 integrin-dependent manner.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9 knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of ß2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.

The interplay of galectins-1, -3, and -9 in the immune-inflammatory response underlying cardiovascular and metabolic disease.

Galectins are ß-galactoside-binding proteins that bind and crosslink molecules via their sugar moieties, forming signaling and adhesion networks involved in cellular communication, differentiation, migration, and survival. Galectins are expressed ubiquitously across immune cells, and their function varies with their tissue-specific and subcellular location. Particularly galectin-1, -3, and -9 are highly expressed by inflammatory cells and are involved in the modulation of several innate and adaptive immune responses. Modulation in the expression of these proteins accompany major processes in cardiovascular diseases and metabolic disorders, such as atherosclerosis, thrombosis, obesity, and diabetes, making them attractive therapeutic targets. In this review we consider the broad cellular activities ascribed to galectin-1, -3, and -9, highlighting those linked to the progression of different inflammatory driven pathologies in the context of cardiovascular and metabolic disease, to better understand their mechanism of action and provide new insights into the design of novel therapeutic strategies.

Galectin-9: A novel promoter of atherosclerosis progression.

BACKGROUND AND AIMS: Atherosclerosis is widely accepted to be an inflammatory disease driven by lipid accumulation and leukocyte recruitment. More recently, galectins, a family of ß-galactoside binding proteins, have been shown to play a role in leukocyte recruitment among other immunomodulatory functions. Galectin (Gal) -9, a tandem repeat type galectin expressed by the endothelium in inflammatory environments, has been proposed to promote leukocyte recruitment. However, the role of Gal-9 in the context of monocyte recruitment remains elusive. METHODS AND RESULTS: Here, we characterise the immunomodulatory role of Gal-9 in context of atherosclerosis. We show that ApoE-/-Gal-9-/- mice have a significantly reduced aortic plaque burden compared to their ApoE-/- littermate controls after 12 weeks of high fat diet. RNA sequencing data from two independent studies reveal Lgals9 expression in leukocyte clusters isolated from murine atherosclerotic plaques. Additionally, soluble Gal-9 protein induces monocyte activation and a pro-inflammatory phenotype in macrophages. Furthermore, we show that immobilised recombinant Gal-9 acts as capture and adhesion molecule for CD14+ monocytes in a ß2-integrin and glycan dependent manner, while adhesion of monocytes to stimulated endothelium is reduced when Gal-9 is knocked down. Gal-9 also facilitates enhanced recruitment of leukocytes from peripheral arterial disease (PAD) patients compared to healthy young and aged controls. We further characterise the endothelium as source of circulating Gal-9, which is increased in plasma of PAD patients compared to healthy controls. CONCLUSIONS: These results highlight a pathological role for Gal-9 as promoter of monocyte recruitment and atherosclerotic plaque progression, making it a novel target in the prevention of plaque formation and progression.

Glycans and Glycan-Binding Proteins as Regulators and Potential Targets in Leukocyte Recruitment.

Leukocyte recruitment is a highly controlled cascade of interactions between proteins expressed by the endothelium and circulating leukocytes. The involvement of glycans and glycan-binding proteins in the leukocyte recruitment cascade has been well-characterised. However, our understanding of these interactions and their regulation has expanded substantially in recent years to include novel lectins and regulatory pathways. In this review, we discuss the role of glycans and glycan-binding proteins, mediating the interactions between endothelium and leukocytes both directly and indirectly. We also highlight recent findings of key enzymes involved in glycosylation which affect leukocyte recruitment. Finally, we investigate the potential of glycans and glycan binding proteins as therapeutic targets to modulate leukocyte recruitment and transmigration in inflammation.

Bcl-2/Bcl-xL inhibitor ABT-263 overcomes hypoxia-driven radioresistence and improves radiotherapy.

Hypoxia, a characteristic of most human solid tumors, is a major obstacle to successful radiotherapy. While moderate acute hypoxia increases cell survival, chronic cycling hypoxia triggers adaptation processes, leading to the clonal selection of hypoxia-tolerant, apoptosis-resistant cancer cells. Our results demonstrate that exposure to acute and adaptation to chronic cycling hypoxia alters the balance of Bcl-2 family proteins in favor of anti-apoptotic family members, thereby elevating the apoptotic threshold and attenuating the success of radiotherapy. Of note, inhibition of Bcl-2 and Bcl-xL by BH3-mimetic ABT-263 enhanced the sensitivity of HCT116 colon cancer and NCI-H460 lung cancer cells to the cytotoxic action of ionizing radiation. Importantly, we observed this effect not only in normoxia, but also in severe hypoxia to a similar or even higher extent. ABT-263 furthermore enhanced the response of xenograft tumors of control and hypoxia-selected NCI-H460 cells to radiotherapy, thereby confirming the beneficial effect of combined treatment in vivo. Targeting the Bcl-2 rheostat with ABT-263, therefore, is a particularly promising approach to overcome radioresistance of cancer cells exposed to acute or chronic hypoxia with intermittent reoxygenation. Moreover, we found intrinsic as well as ABT-263- and irradiation-induced regulation of Bcl-2 family members to determine therapy sensitivity. In this context, we identified Mcl-1 as a resistance factor that interfered with apoptosis induction by ABT-263, ionizing radiation, and combinatorial treatment. Collectively, our findings provide novel insights into the molecular determinants of hypoxia-mediated resistance to apoptosis and radiotherapy and a rationale for future therapies of hypoxic and hypoxia-selected tumor cell fractions.

Characterisation of endogenous Galectin-1 and -9 expression in monocyte and macrophage subsets under resting and inflammatory conditions.

Macrophages are key cells in both acute and chronic inflammatory settings. Their activation and function highly depends on the cytokines, chemokines and adhesion molecules that direct monocytes to infiltrate tissues, differentiate into macrophages, and finally lead to the clearance of such inflammatory signals. Galectins, ß-galactoside-binding lectins, are differentially expressed by various immune cells, and some members of this family have been identified as regulators of leukocyte recruitment and activation. Galectin-1 (Gal-1) and galectin-9 (Gal-9) expression has been described in immune cells, but the specific molecular mechanisms by which they modulate the inflammatory response in macrophages/monocytes are not completely understood. In this study we sought to comprehensively characterise the expression profile of endogenous Gal-1 and Gal-9 in different murine and human monocyte/macrophage populations in response to different inflammatory stimuli. All subsets of murine and human macrophages expressed significant levels of Gal-1 and -9. Interestingly, murine bone marrow derived macrophages stimulated with M2 (pro-resolution) polarising agents preferentially upregulated Gal-1, while Gal-9 expression was upregulated by M1/pro-inflammatory stimulation. However, we observed differing results in human monocyte derived macrophages. Collectively, our findings report a differential expression pattern of endogenous Gal-1 and -9 in macrophage and monocyte subsets in response to a range of inflammatory stimuli. Future studies will endeavour to elucidate whether the galectins make attractive therapeutic targets or agents for regulating the inflammatory response.

Triggering the Resolution of Immune Mediated Inflammatory Diseases: Can Targeting Leukocyte Migration Be the Answer?

Leukocyte recruitment is a pivotal process in the regulation and resolution of an inflammatory episode. It is vital for the protective responses to microbial infection and tissue damage, but is the unwanted reaction contributing to pathology in many immune mediated inflammatory diseases (IMIDs). Indeed, it is now recognized that patients with IMIDs have defects in at least one, if not multiple, check-points regulating the entry and exit of leukocytes from the inflamed site. In this review, we will explore our understanding of the imbalance in recruitment that permits the accumulation and persistence of leukocytes in IMIDs. We will highlight old and novel pharmacological tools targeting these processes in an attempt to trigger resolution of the inflammatory response. In this context, we will focus on cytokines, chemokines, known pro-resolving lipid mediators and potential novel lipids (e.g., sphingosine-1-phosphate), along with the actions of glucocorticoids mediated by 11-beta hydroxysteroid dehydrogenase 1 and 2.