Using super-resolution microscopy to study immune cell biology in asthma and chronic obstructive pulmonary disease

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The key focus of my research over recent years has been in addressing important problems in cell biology and immunology with novel and state-of-the-art photonics technology combined with molecular and cell biology techniques. Our imaging studies have helped establish an emerging new paradigm that interactions between immune cell receptors, kinases and adaptors are at least in part controlled by the dynamics of supramolecular assemblies at an intercellular contact or immune synapse. Thus, immune cell recognition and signalling is governed by transient interactions between heterogeneous clusters of proteins, a substantially different concept from the linear cascade of individual protein-protein interactions depicted in textbooks. From these data, emerges a new hypothesis; that the nanoscale organisation of immune cell surfaces varies in health and disease which this significantly impacts immunity. This idea has the potential to seed an entirely novel route to drug design; manipulating the organisation of the immune cell surface to augment or dampen immune cell responses. A considerable body of preliminary data underpins this specific proposal. It has been assumed that activating Fc receptors and the inhibitory receptor signal regulatory protein alpha (SIRPalpha) are evenly distributed at the macrophage cell surface and cluster upon ligation or cross-linking. However, we have recently found that these proteins are not homogeneously distributed at the cell surface but are organized in nanometre-scale clusters at the surface of primary human macrophages (manuscript pending revisions). In resting cells, nanoclusters of activating and inhibitory receptors are co-localized, constrained by the actin cytoskeleton. Ligation of Fc receptor, and subsequent signaling via Src-family kinases, induces the segregation of activating and inhibitory nanoclusters, establishing a positive feedback for cellular activation, by segregating phosphatase and kinase activity; a process blocked by co-ligation of inhibitory receptors.We now aim to build upon these findings to explore how the changing arrangement of cell surface proteins impacts disease. Using alveolar macrophages taken from healthy donors or patients, we will compare, for example, the extent to which SIRPalpha inhibits Fc receptor signaling in comparison to its proximity to the activating Fc receptors which mediate phagocytosis, as well as signalling events from these activating receptors. This will reveal how an altered organisation of macrophage surfaces could impact impaired pathogen recognition or resolution of inflammation in the lung. We will use patterned surfaces to further test the importance of the proximity of activating and inhibitory signals in signal integration. These data will establish how changes in the cell surface organisation impacts disease states in lung inflammation.