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

近年来，我的研究重点一直是通过新颖和最先进的光子学技术与分子和细胞生物学技术相结合来解决细胞生物学和免疫学中的重要问题。我们的成像研究帮助建立了一个新兴的新范式，即免疫细胞受体、激酶和适配器之间的相互作用至少部分受细胞间接触或免疫突触的超分子组装动力学控制。因此，免疫细胞识别和信号传递是由不同蛋白质簇之间的瞬时相互作用控制的，这是一个与教科书中描述的单个蛋白质-蛋白质相互作用的线性级联本质上不同的概念。从这些数据中，出现了一个新的假设；免疫细胞表面的纳米级组织在健康和疾病中有所不同，这显著影响了免疫力。这一想法有可能为药物设计开辟一条全新的途径；操纵免疫细胞表面的组织来增强或抑制免疫细胞的反应。大量的初步数据支持了这一具体提议。激活的Fc受体和抑制性受体信号调节蛋白α(SIRPalpha)被认为是均匀分布在巨噬细胞表面，并在连接或交联时聚集在一起。然而，我们最近发现，这些蛋白质并不是均匀分布在细胞表面，而是在原代人类巨噬细胞表面以纳米级的簇组织起来(手稿有待修改)。在静息细胞中，激活和抑制受体的纳米簇是共定位的，受到肌动蛋白细胞骨架的限制。Fc受体的连接，以及随后通过Src家族激酶的信号传递，导致激活和抑制纳米簇的分离，通过分离磷酸酶和激酶活性，为细胞激活建立正反馈；这一过程被抑制受体的共同连接所阻断。我们现在的目标是在这些发现的基础上，探索细胞表面蛋白排列的变化如何影响疾病。使用取自健康供者或患者的肺泡巨噬细胞，我们将比较，例如，SIRPalpha抑制Fc受体信号的程度与其与介导吞噬作用的激活Fc受体的接近程度，以及来自这些激活受体的信号事件。这将揭示巨噬细胞表面组织的改变如何影响肺部炎症的病原体识别或分解。我们将使用图案化的表面进一步测试激活和抑制信号在信号整合中的接近程度的重要性。这些数据将确定细胞表面组织的变化如何影响肺部炎症的疾病状态。

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.