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

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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.