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Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice
Published on: March 24, 2015
Cholesterol oxidase treatment impairs CXCR4-mediated T cell migration
Sofía R Gardeta1, Eva M García-Cuesta1, Blanca Soler Palacios1
1Chemokine Signaling group, Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, Madrid, E-28049, Spain.
Background:
Cholesterol, a key component of mammalian cell membranes, modulates the properties of the lipid bilayer and influences the conformational states of membrane receptors, including G protein-coupled receptors (GPCRs). These effects are mediated through direct interactions with specific residues within the transmembrane regions and modulation of the surrounding lipid bilayer. Chemokine receptors, a GPCR sub-family, adopt distinct conformations associated with specific cell functions. For example, CXCL12 triggers receptor clustering, essential for directional cell migration. However, the precise mechanisms by which cholesterol controls the spatial organization of these receptors remain unclear. This study investigated the role of cholesterol in modulating the chemokine receptor CXCR4.
Methods:
We used lipidomic analysis to measure cellular cholesterol levels, and raster image correlation spectroscopy to assess the impact of cholesterol depletion on membrane fluidity. CXCR4 nanoclustering and dynamics were examined using single-particle tracking in TIRF mode. CXCR4 dimer formation was evaluated by FRET and FLIM analyses, and directed cell migration was measured using microfluidic chemotaxis chambers. Receptor expression and ligand binding were determined by flow cytometry with specific antibodies and CXCL12-ATTO700. Additional assays included calcium flux, and western blotting for signaling molecules. Statistical analysis used unpaired t-tests, one-way ANOVA, and two-tailed Mann-Whitney tests.
Results:
Our findings demonstrate that moderate cholesterol depletion using cholesterol oxidase increases membrane fluidity, impairs T cell migration towards CXCL12 gradients, and enhances CXCL12-mediated β1-integrin activation. This treatment also induced alterations in CXCR4 conformation and spatial distribution, without significantly affecting ligand binding or other chemokine-mediated signaling pathways. Immunocytochemical analysis indicated that cholesterol oxidase primarily affected the largest CXCR4 clusters, with no significant impact on lipid-enriched microdomains.
Conclusions:
This study identifies cholesterol as a crucial regulator of CXCR4 lateral mobility and spatial organization, enabling cells to effectively sense chemoattractant gradients.
