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

硬皮病(SSC)是一种典型的纤维性疾病，自身免疫介导的损伤通过细胞外基质在皮肤和内脏的积聚导致组织功能的丧失。SSC中活化的成纤维细胞作为抗原提呈细胞(APC)，启动或维持自身免疫和组织损伤。然而，激活的免疫细胞对APC的影响(“反向信号传递”)及其对纤维化的贡献才刚刚开始被认识。SSC中成纤维细胞激活的最终触发因素尚不完全清楚，但它似乎是由调节失调的形态原信号通路：Hedgehog、转化生长因子β和Wnt的作用形成的。如上所述，激活的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配体表达细胞的变化，称为反向信号传递。本博士项目将研究从SSC患者或健康对照组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因子对成纤维细胞和内皮细胞的作用机制和相互作用。这些将是研究的起点，但具体目标将在分析第一年获得的先导数据后确定：1-调查共刺激激动型抗体对Shh、Wnt3a、Wnt5A、Wnt10A和TGFb细胞反应性的影响；2-在共刺激配体参与的情况下对成纤维细胞中基因表达的变化进行RNA-SEQ分析。3-确定(1)中所示的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、选定基因的过度表达和沉默(SiRNA)、免疫沉淀和质谱学鉴定相互作用蛋白、CRISPR-Cas9基因组编辑功能分析以研究Shh、Wnt和TGFb信号转导、qRT-PCR、Western印迹。

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.