The diverse origins, nanometre-scale and invasive isolation procedures associated with extracellular vesicles (EVs) mean they are usually studied in bulk and disconnected from their parental cell. Here, we used super-resolution microscopy to directly compare EVs secreted by individual human monocyte-derived macrophages (MDMs). MDMs were differentiated to be M0-, M1-or M2-like, with all three secreting EVs at similar densities following activation. However, M0-like cells secreted larger EVs than M1-and M2-like macrophages. Proteomic analysis revealed variations in the contents of differently sized EVs as well as between EVs secreted by different MDM phenotypes. Super resolution microscopy of single-cell secretions identified that the class II MHC protein, HLA-DR, was expressed on ∼40% of EVs secreted from M1-like MDMs, which was double the frequency observed for M0-like and M2like EVs. Strikingly, human macrophages, isolated from the resected lungs of cancer patients, secreted EVs that expressed HLA-DR at double the frequency and with greater intensity than M1-like EVs. Quantitative analysis of single-cell EV profiles from all four macrophage phenotypes revealed distinct secretion types, five of which were consistent across multiple sample cohorts. A sub-population of M1-like MDMs secreted EVs similar to lung macrophages, suggesting an expansion or recruitment of cells with a specific EV secretion profile within the lungs of cancer patients. Thus, quantitative analysis of EV heterogeneity can be used for single cell profiling and to reveal novel macrophage biology.

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 5 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.

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.