Geometric control of macrophage phenotype and function: a single cell analysis

Macrophages are tissue-resident immune cells that play critical roles in development, metabolic regulation, maintenance of tissue homeostasis and defense against invading pathogens. To carry out their diverse functional roles, Macrophages rely on their remarkable plasticity to assume a continuum of different functional phenotypes in response to their ever changing microenvironment. Precise spatiotemporal regulation of macrophage phenotype is necessary as any abnormalities could lead to chronic inflammation, the hallmark of many diseases such as cancer and atherosclerosis. While it is thought that biochemical signals such as cytokines and chemokines are the primary regulators of macrophage phenotype polarization, evidence has begun to emerge that tissue architecture and physical cues in the extracellular microenvironment can also contribute to their phenotype and function. Here, we show that macrophage cell shape can similarly provide an instructive signal for macrophage phenotype polarization.One of the major challenges in studying macrophages is to accurately identify phenotypic subsets within a heterogeneous population. Traditional bulk measurements of cellular attributes, such as ELISA, Western blot and PCR, can overlook these heterogeneities and the potential role of rare subsets in initiating an immune response. Furthermore, the effects of complex microenvironmental cues, both soluble and physical, on macrophage activation may be lost when only the average response of a cell population is examined. In order to tease apart the nuanced contributors to the functional variances among macrophages, single cell analyses of phenotype and function must be utilized. While flow cytometry combined with immunofluorescence and fluorescence in situ hybridization (FISH) has already been used in phenotying and genotyping single cells, functional assessment through secreted products remain challenging. Recent chip-based technologies have used micro-/nano- fabricated wells to physically isolate single T cells and B cells and examined their secreted products. However, these are non-adherent cells that are less sensitive to the physical and adhesive cues provided by the extracellular matrix. To examine adherent cells such as macrophages, we herein created a single cell cytokine detection platform that allows for controlled physical and adhesive microenvironment. We used this platform to examine single macrophage cytokine secretion under different soluble factors, on different adhesive substrates, and with different cell shape. This work broadens our understanding of macrophage activation in the context of pathophysiological conditions. It further demonstrates the importance of physical and adhesive cues on macrophage activation and function, and the necessity to include these factors when studying macrophages in vitro. Our novel device allows precise control of macrophage adhesion and simultaneous detection of secreted products on a single cell level, which will facilitate future research on this topic