Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells

Abstract

Intracellular vesicle transport is essential to maintain cellular homeostasis and is known to be mediated by the endosomal pathway and SNARE proteins. Endosomal-mediated transport to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of effector immune responses. Despite the critical importance of this process, the specific molecular agents that regulate DC cytokine secretion are poorly characterised. Galectin-9, a ß-galactoside-binding protein, has emerged as novel modulator of numerous cellular processes although its exact functions remain to be elucidated. In particular, its intracellular roles in regulating (immune) cell homeostasis and cytosolic transport are virtually unknown. Here, we investigated galectin-9 function in cytokine secretion in primary human DCs, and report that galectin-9 is essential for intracellular trafficking of cytokines to the cell surface. Galectin-9 depletion resulted in the accumulation of cytokine-containing vesicles in the Golgi complex that eventually underwent lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using unbiased immunoprecipitation-mass spectrometry technology, and identified that galectin-9 was required for rerouting of Vamp-3-containing endosomes to the plasma membrane for cargo release upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle machinery and transport and shed new light into the multiple roles galectin and glycan-mediated interactions play in governing cell function.

Galectin-9 regulates dendritic cell contractility and migration via RhoA

“…Illustrating its versatility, galectin-9 was first characterised as an eosinophil chemo-attractant 23 , it induces cell death and immune tolerance by binding T cell immunoglobulin-3 in T helper 1 (TH1) cells 24 , it promotes the expansion of immunosuppressive macrophages 25 and monocytic myeloid-derived suppressor cells 26 and it has been shown to negatively regulate B cell receptor signalling 27,28 . Contrary to these reports implicating an immunosuppressive role for galectin-9, we and others have previously identified galectin-9 to positively regulate DC biology although many of its functions in myeloid cells remain poorly defined [29][30][31] .…”

“…We next set to unravel the molecular mechanisms by which membrane-bound galectin-9 modulates RhoA activity at the cytosol thereby regulating actin polymerisation and contractility in DCs. The transmembrane adhesion protein CD44 has been shown to interact with galectin-9 in T cells and Natural Killer cells 48,49 and we identified CD44 to be an interacting galectin-9 binding partner in moDCs 29 . Furthermore, CD44 interacts with RhoA via its cytoplasmic tail and its spatial organisation is determined by the dynamic remodelling of the actin cytoskeleton.…”

“…A total of 15 μg siRNA (5 μg from each siRNA) was transferred to a 4-mm cuvette (Bio-Rad) and 5-10x10 6 DCs were added in 200 μl OptiMEM and incubated for 3 min before being pulsed with an exponential decay pulse at 300 V, 150 mF, in a Genepulser Xcell (Bio-Rad, Veenendaal, Netherlands), as previously described 29,30 . Immediately after electroporation, cells were transferred to preheated (37 °C) phenol red-free RPMI 1640 culture medium supplemented with 1 % ultraglutamine, 10 % (v/v) FCS, IL-4 (300 U/ml), and GM-CSF (450 U/ml) and seeded at a final density of 5x10 5 cells/ml.…”

Galectokines: The Promiscuous Relationship between Galectins and Cytokines

“…In addition, it was recently reported that galectin-9 facilitates the trafficking of cytokines to the cell surface in dendritic cells. Galectin-9 depletion resulted in the accumulation of cytokine-containing vesicles in the Golgi complex that eventually underwent lysosomal degradation [ 116 ].…”

Galectins in Esophageal Cancer: Current Knowledge and Future Perspectives

“…For example, in the cytoplasm galectin-1 dimers can directly interact with Raf-effectors which subsequently improves H-Ras nanoclustering in the membrane [ 30 ]. Recently, galectin-9 was suggested to interact with VAMP-3 in order to control cytokine trafficking in dendritic cells [ 31 ]. Haudek et al provided an extensive overview of the intracellular binding partners of galectin-3, which includes nuclear proteins involved in splicing [ 32 ].…”