Identifying therapeutic targets for human chronic wounds using single cell transcriptomics and digital spatial profiling

由于人口老龄化和包括糖尿病和肥胖症在内的风险因素的患病率增加，慢性皮肤伤口是一个主要且不断增加的临床负担。这些伤口在身体和心理上使人衰弱，感染和复发的风险很高，是截肢的最常见原因，5年死亡率为45%（相当于结肠癌）。尽管疾病负担如此沉重，但以科学为主导的治疗方法却极为缺乏。这在一定程度上是由于缺乏基于精确医学的方法，缺乏探索人类疾病与临床前模型之间联系的比较数据。 巨噬细胞（MF）是表型可塑性细胞，涉及支持“正常”修复过程和在异常愈合情况下发挥有害作用。先前的研究表明，MF是功能失调的，在小鼠和人的皮肤愈合受损的情况下表现出不受限制的激活。因此，MF代表了慢性伤口的有吸引力的治疗靶标。然而，MFs是高度异质性和动态的细胞，使得识别特定的致病和促修复亚群具有挑战性。我们的目标是提供一个更系统的观点，在急性和慢性伤口皮肤伤口修复的动态过程中，在人类和小鼠模型，我们的实验室建立，中心的作用，巨噬细胞。为了规避这一点，我们目前正在两个主要项目中使用无偏倚的单细胞RNA测序方法：1）定义小鼠急性皮肤伤口中的促修复MF群体，2）在我们的新型人类慢性伤口临床前模型中鉴定致病性MF群体。我们还进行了Nanostring数字空间分析（DSP），以确定人类慢性伤口的免疫环境，并支持识别皮肤溃疡形成和持续性中的"核心"途径。该项目的初始阶段将侧重于通过DSP，结合多参数流式细胞术，组织学和成像，从人类急性伤口和慢性皮肤溃疡中分离MFs亚群。啮齿动物皮肤伤口中的MF反映了在人类慢性伤口中鉴定的那些特征的许多特征。然而，小鼠临床前模型和人类慢性皮肤伤口之间的精确推论亚群尚未确定。我们将建立一种方法来整合来自人类急性和慢性皮肤创伤的数据，包括单细胞RNA测序和DSP，我们将解析促修复和致溃疡MF亚群。我们将继续使用最先进的计算方法将这些细胞的转录组映射到目前在我们的小鼠急性和慢性伤口模型中鉴定的转录组。该项目将能够识别调节皮肤溃疡形成和持续性与跨物种有效伤口愈合的“核心”途径，从而定义慢性皮肤伤口的易处理治疗靶点。我们将在我们的小鼠模型上进行干预研究，以在后期验证这些抗溃疡治疗方法。该项目将是爱丁堡大学和格拉斯哥大学之间的合作努力。它将能够培训尖端的计算分析技能以及体内和体外湿实验室技术。

Chronic skin wounds are a major and increasing clinical burden as a result of an aging population and increased prevalence of risk factors including diabetes and obesity. These wounds are physically and psychologically debilitating, have a high risk of infection and reoccurrence, are the commonest cause of limb amputation and have a 5 year mortality rate of 45% (equivalent to colon cancer). Despite this significant burden of disease there is a profound paucity of science-led therapeutics. This is in part due to the lack of a precision medicine-based approach, with a dearth of comparative data exploring the links between human disease and pre-clinical models. Macrophages (MFs) are phenotypically plastic cells implicated both in supporting the 'normal' repair process and in playing detrimental roles in aberrant healing scenarios. Previous studies indicate that MFs are dysfunctional, exhibiting unrestrained activation in the context of impaired skin healing in both mouse and human. MFs therefore represent an attractive therapeutic target for chronic wounds. However, MFs are highly heterogeneous and dynamic cells, making it challenging to identify specific pathogenic and pro-repair subpopulations. We aim to provide a more systematic view on the dynamic process of skin wound repair in both acute and chronic wounds, and in both human and the mouse model our lab established, centring the role of macrophages. To circumvent this, we are currently using an unbiased single-cell RNA-sequencing approach in two major projects: 1) defining the pro-repair MF population in murine acute skin wounds and 2) identifying pathogenic MF populations in our novel pre-clinical model of human chronic wounds. We have also performed Nanostring Digital Spatial Profiling (DSP) to define the immunological milieu of human chronic wounds and support the identification of "core" pathways in skin ulcer formation and persistence. The initial phase of the project will focus on resolving MFs subpopulations from human acute wounds and chronic skin ulcers by DSP, coupled with multiparameter flow cytometry, histology and imaging. MFs in rodent skin wounds mirror a number of features of those identified in human chronic wounds. However, the precise corollary subpopulations between mouse pre-clinical models and human chronic skin wounds have not yet been defined. We will establish a method to integrate data from human acute and chronic skin wounds, comprising single-cell RNA-sequencing and DSP, we will resolve the pro-repair and ulcerogenic MF subpopulations. We will proceed to map the transcriptomes of these cells to those currently being identified in our mouse acute and chronic wound models using cutting edge computational approaches. This project will enable the identification of 'core' pathways regulating skin ulcer formation and persistence versus effective wound healing across species and thus allow the definition of tractable therapeutic targets for chronic skin wounds. We will perform intervention studies on our mouse models to validate these anti-ulcerogenic therapeutic approaches at a later stage. This project will be a collaborative effort between University of Edinburgh and University of Glasgow. It will enable the training of both cutting-edge computational analysis skills as well as in-vivo and in-vitro wet lab techniques.