Activation-induced cell protrusions and vesicle secretion of human lung macrophages revealed by super-resolution microscopy

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Abstract Recent studies using super-resolution microscopy have established that the organisation of immune cell receptors impacts signal integration and cellular activation. Understanding the nano-scale dynamics of surface receptors on tissue specific macrophages is especially important, as they are phenotypically diverse and possess a large repertoire of receptors. However, tissue macrophages are highly auto-fluorescent, severely limiting the utility of light microscopy. Here, we report a novel correction technique which utilises a moving median filter to remove auto-fluorescent noise from stochastic optical reconstruction microscopy datasets. Using this, we visualised lung macrophages (LMs) activated through Fc receptors by IgG-coated glass slides, representing a 2D model of the phagocytic synapse. This unexpectedly revealed the formation of protrusions, at the surface of LMs but not blood-derived macrophages. Class I MHC protein accumulated at the tips, consistent with a role for macrophage protrusions in antigen presentation. Additionally, staining for the exosome marker CD81 revealed the secretion of extracellular vesicles. Classically, cell-derived vesicles are studied after bulk isolation, which is harsh and comes with many caveats. Imaging them directly upon secretion allows analysis of their properties on a cell-by-cell basis in a near-native state. We discovered that LM vesicles appeared distinct from those secreted from blood-derived macrophages in that their average diameter was much smaller (80 nm ± 19 nm vs. 159 nm ± 78 nm). Thus, our correction method for super-resolution microscopy revealed novel cell biology – protrusion formation and vesicle secretion – triggered upon activation of human LMs.