Investigating interplay between morphogen and immune signalling in the development of fibrosis

硬皮病（SSc）是一种典型的纤维化疾病，其自身免疫介导的损伤通过在皮肤和内脏器官中积累细胞外基质导致组织功能丧失。已知SSc中活化的成纤维细胞作为抗原呈递细胞（apc）启动或延续自身免疫和组织损伤。然而，激活免疫细胞对apc（“反向信号传导”）的影响及其对纤维化的贡献才刚刚开始得到重视。SSc中成纤维细胞激活的最终触发因素尚不完全清楚，但它似乎是由失调的形态因子信号通路形成的：Hedgehog，转化生长因子β和Wnt。如上所述，活化的SSc成纤维细胞表达共刺激配体来启动免疫系统，但共刺激配体与T细胞的结合也会引起表达配体的细胞发生变化，称为“反向信号传导”。这个博士项目将研究从SSc患者或健康对照中分离的成纤维细胞和内皮细胞中选择的共刺激配体和形态原的机制和串扰。这些将是研究的起点，但具体目标将在第一年获得的试点数据分析后确定：1-研究共刺激激动抗体对细胞对shh、Wnt3A、Wnt5A、Wnt10A和TGFb的反应性的影响。2-在共刺激配体参与下，对成纤维细胞的基因表达变化进行RNA-seq分析。确定(1)中形态因子对共刺激配体表达的影响。实验方法：细胞培养，RNA-seq分析，选定基因的过表达和沉默（siRNA），免疫沉淀和质谱鉴定相互作用蛋白，CRISPR-Cas9基因组编辑，功能分析研究Shh， Wnt和TGFb信号，qRTPCR, western blot。

Scleroderma (SSc) is a prototypic fibrotic disease, in which an autoimmune-mediated injury leads to loss of tissue function through accumulation of extracellular matrix in the skin and internal organs. Activated fibroblasts in SSc are known to act as antigen presenting cells (APCs) to initiate or perpetuate autoimmunity and tissue damage. However, the effects of activated immune cells on APCs ("reverse signalling"), and its contribution to fibrosis, is just starting to be appreciated.The definitive trigger of fibroblast activation in SSc is not fully understood but it seems to be shaped by a contribution of dysregulated morphogen signalling pathways: Hedgehog, Transforming Growth Factor Beta and Wnt. As mentioned above, activated SSc fibroblasts express co-stimulatory ligands to prime the immune system, but engagement of the co-stimulatory ligands with T cells also elicits changes in the ligand-expressing cells, called "reverse signalling".This PhD project will investigate the mechanisms and crosstalk of selected co-stimulatory ligands and morphogens on fibroblasts isolated from SSc patients or healthy controls and in endothelial cells. These will be the starting lines of investigation, but specific aims will be defined after analysis ofpilot data obtained during the first year:1- Investigate the effect of co-stimulatory agonistic antibodies on the cells responsiveness toShh, Wnt3A, Wnt5A, Wnt10A, and TGFb.2- Perform RNA-seq analysis of gene expression changes in fibroblasts in the presence of costimulatoryligand engagement.3- Determine the effect of morphogens indicated in (1) on expression of co-stimulatory ligands.Experimental approach: Cell culture, RNA-seq analysis, overexpression and silencing (siRNA) of selected genes, identification of interacting proteins by immunoprecipitation and mass spectrometry, genome editing by CRISPR-Cas9, functional assay to study Shh, Wnt and TGFb signalling, qRTPCR, western blot.